TWI614264B - Peptide core-based multi-arm linkers and their applications - Google Patents

Abstract

This disclosure proposes a variety of molecular constructs that have a target element and an effector element. Methods of using the molecular constructs to treat a variety of diseases are also disclosed herein.

Description

Multi-arm conjugate containing peptide core and application thereof

The present disclosure relates to the field of pharmacy; more specifically, it relates to multifunctional molecular constructs, such as molecular constructs having target elements and effector elements to deliver effector elements (such as therapeutic drugs) to the target site.

In recent years, a variety of screening and selection methods for monoclonal antibodies (mAbs) with antigens as targets have been developed in the field, which has led to the development of many therapeutic antibodies, which have recently been considered incurable. The disease brings dawn. According to the Therapeutic Antibody Database, as many as 2,800 antibodies have been or will be put into human clinical trials, while about 80 antibodies are approved by the government's drug regulatory agency for clinical treatment. From the large amount of data related to the efficacy of antibodies, it is known which pharmacological mechanisms the antibodies use to exert their effects.

When antibodies are used as therapeutic agents, the main pharmacological mechanism is the use of antibodies to neutralize or trap the mediators that cause disease; the mediators may be cytokines in the blood circulation, interstitial space or lymph nodes or Immune components. Neutralizing the activity of the mediator can inhibit the interaction between the mediator causing the disease and its receptor. In addition, the soluble receptor of the cytokine or the extracellular part of the receptor and the Fc part of the immunoglobulin IgG can be made into a fusion protein. Such a fusion protein can neutralize the cytokine by using a mechanism similar to a neutralizing antibody. Or immune factors; therefore, many fusion protein therapeutics have also been developed.

In addition, some therapeutic antibodies that are licensed for clinical use or are undergoing clinical research use a mechanism different from the above-mentioned main antibody mechanisms to exert their pharmacological effects, such as blocking these receptors by binding to receptors And its ligands. For such antibody drugs, the main pharmacological mechanism is not Fc-mediated (such as antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytolysis , CMC), etc.).

Other therapeutic antibodies can bind to certain surface antigens on target cells and thereby exert Fc-mediated functions and other mechanisms on target cells. The most important Fc-mediated mechanisms are antibody-dependent cytotoxicity (ADCC) and complement-mediated cytolysis (CMC), both of which will lyse target cells that bind to the antibody. When these antibodies bind to specific cell surface antigens, they can cause apoptosis in the target cells they bind to.

The concept and method of preparing antibodies with dual specificity has sprouted as early as 30 years ago. In recent years, with the advancement of recombinant antibody engineering methods and the demand for better drugs, a variety of bispecific antibodies with different structural configurations have been developed.

For example, a bivalent or multivalent antibody may carry two or more antigen-binding sites. A variety of methods are known for preparing multivalent antibodies, and these methods usually use three linking structures to covalently link three or four antigen-binding fragments (Fabs) together. For example, antibodies have been made through recombinant engineering that can express three or four tandem Fab repeats.

In addition, a method of manufacturing a multivalent antibody by using a synthetic crosslinker to chemically connect different antibodies or binding sites together is also a technique known in the art. One way is to use three different linkers to join three, four or more separated Fab fragments to each other through chemical cross-linking. Another approach is to first prepare a construct with multiple Fabs and assemble these Fabs into a one-dimensional DNA scaffold. These various multivalent antibody constructs designed to bind to the target molecule differ in size, half-life, conformal elasticity, and ability to regulate the immune system.

Based on the above description, some studies have focused on the preparation of molecular constructs with a fixed number of effector elements or molecular constructs with two or more different functional elements, such as at least one target element and at least one effect element. However, it is often difficult to use chemical synthesis or recombination techniques to build molecular constructs with specific targets combined with effector elements. Therefore, there is an urgent need in the art for a novel molecular platform that can be used to construct a variety of molecules suitable for a variety of diseases.

This summary is intended to provide a simplified summary of this disclosure so that readers may have a basic understanding of this disclosure. This summary is not a comprehensive overview of the disclosure, and it is not intended to indicate important / critical elements of the specific embodiments of the invention or to define the scope of the invention. The sole purpose of the summary is to provide a simplified description of certain concepts disclosed herein to facilitate an understanding of the embodiments described later.

＜ I ＞ Multiarm conjugate with peptide core

A first aspect of the present disclosure relates to a bonding unit to which at least two different functional elements are connected. For example, the junction unit can be connected with two different effect elements, a target element and an effect element, or an effect element and a polyethylene glycol (PEG) chain that can extend the life cycle of the junction unit. . The bonding unit of the present invention is designed to carry at least two different functional groups so that these functional elements can react with the respective functional groups to be connected to the bonding unit. Therefore, the junction unit of the present invention can be used as a platform for preparing a molecular construct with two or more functional elements.

According to various embodiments of the present disclosure, the bonding unit includes a central core and a plurality of connecting arms. The central core may be a polypeptide core, which contains 2 to 15 lysine (K) residues, wherein each K residue and the next K residue are separated by a stuffing sequence, the stuffing sequence Comprising glycine (G) and serine (S) residues; or the sequence of the polypeptide core is (X _aa -K) _n , where X _aa has 2 to 12 ethylene dimers A polyethylene glycol (EG) repeating unit is a PEGylated amino acid, and n is an integer from 2 to 15. In an embodiment implemented as needed, the stuffing sequence is composed of 2 to 20 amino acid residues. In various embodiments, the stuffing sequence may have any of the following sequences: GS, GGS, GSG or sequence number: the sequence shown in 1-16. According to some embodiments of the present disclosure, the central core contains a G _1-5 SK sequence of 2 to 15 units; in a preferred case, the central core has a sequence of (GSK) _2-15 . Each linking arm is connected to the K residue of the central core by forming a amide bond with the K residue. The free end of the linking arm (that is, the end that is not connected to the central core) has a maleimide group, an N-hydroxysuccinimidyl (NHS) group, and an azido group ( azide group, alkyne group, tetrazine group, cyclooctene group, or cyclooctyne group. In addition, the amino acid residue at the N- or C-terminus of the central core has an azide or alkynyl group; or, or in addition, the amino acid residue at the N- or C-terminus of the central core is cysteine (C) residue, and a coupling arm is connected to the cysteine residue through the sulfhydryl group of the cysteine residue. At this time, the free end of the coupling arm not connected to the central core has an azide group, an alkynyl group, and a tetrazine Or cyclooctenyl or cyclooctynyl.

According to some embodiments of the present disclosure, when the free end of the linking arm is azide, alkynyl, or cyclooctynyl, the amino acid residue at the N- or C-terminus of the central core is a cysteine residue And the free end of the coupling arm is tetrazinyl or cyclooctenyl. According to other embodiments of the present disclosure, when the free end of the linking arm is a tetrazinyl or cyclooctenyl group, the amino acid residue at the N- or C-terminus of the central core has the azide or alkynyl group; or The amino acid residues at the N- or C-terminus of the central core are cysteine residues, and the free ends of the coupling arms are azide, alkynyl, or cyclooctynyl.

In some embodiments, the linking arm is a PEG chain; in a preferred case, it has 2 to 20 EG repeat units. Alternatively, the linking arm is a PEG chain having 2 to 20 EG repeat units and has a double sulfur bond at its free end (ie, the end not connected to the central nuclear K residue). In some embodiments, the coupling arm is a PEG chain; in a preferred case, it has 2 to 12 EG repeat units.

Examples of amino acid residues bearing azido groups include: L-azidohomoalanine (AHA), 4-azido-L-phenylalanine, 4-azido -D-phenylalanine (4-azido-D-phenylalanine), 3-azido-L-alanine (3-azido-L-alanine), 3-azido-D-alanine (3-azido-D- alanine), 4-azido-L-homoalanine, 4-azido-D-homoalanine, 5-azido-D-homoalanine, 5-azido-L- 5-azido-L-ornithine, 5-azido-D-ornithine, 6-azido-L-lysine lysine) and 6-azido-D-lysine. Exemplary residues with alkynylamino acids include: L-homopropargylglycine (L-HPG), D-homopropargylglycine (D-HPG) ) And β-homopropargylglycine (β-HPG).

When the amino acid residue at the N- or C-terminus of the central core is a cysteine residue, the cyclooctenyl group at the free end of the coupling arm may be a trans-cyclooctene (TCO) group, The cyclooctynyl group at the free end of the coupling arm can be dibenzocyclooctyne (DBCO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), or diphenyl Dibenzocyclooctyne (DICO) group. On the other hand, the tetrazinyl group at the free end of the coupling arm includes, but is not limited to: 1,2,3,4-tetrazine, 1,2,3,5-tetrazine, and 1,2,4,5-tetrazine And its derivatives, such as 6-methyltetrazinyl.

According to various embodiments of the present disclosure, the bonding unit further includes a plurality of first elements. In some embodiments, each first element is connected to the connecting arm by forming a amide bond between the connecting arm and the first element. In other embodiments, the "copper catalyzed azide-alkyne cycloaddition (CuAAC)", "stress-assisted azide", -Strain-promoted azide-alkyne click chemistry (SPAAC) reaction or "inverse electron demand Diels-Alder (iEDDA)" reaction, and each first element Connect to the connecting arm.

Where necessary, the bonding unit proposed herein further includes a plurality of engagement arms, which can be individually connected to the connection arms through CuAAC, SPAAC, or iEDDA reactions. According to the embodiment of the present invention, one end of each linking arm for connecting the element (ie, the end not connected to the linking arm) has a cis-butenylimide group or an NHS group. Therefore, each of the first elements and the linking arms can be connected to each other through a sulfide-cis-butene diimide reaction or by forming a amide bond. In some embodiments, each adapter arm is a PEG chain, which preferably has 2 to 20 EG repeat units. In other embodiments, each adapter arm is a PEG chain having 2 to 20 EG repeat units, and the element connecting end thereof has a double sulfur bond.

According to various embodiments implemented as required in the present disclosure, the first element is an effect element capable of exerting a desired effect (eg, a therapeutic effect) in an individual; or, the first element is capable of guiding the joint unit to a target site. Underlying elements. According to an embodiment of the present invention, the first element is fingolimod, fingolimod phosphate, interferon-β, or integrin-α4, β-amyloid, viral protein or bacterial protein specific single-chain variable fragment (scFv).

Also when necessary, the bonding unit may further include a second element different from the first element. In some embodiments, the second element bears an azide group or an alkynyl group, so that it can perform a CuAAC reaction with the corresponding alkynyl group or azide group of the central core or the coupling arm, and is further coupled to the central core or the coupling arm. Or, in some embodiments, the second element is provided with an azido group or a cyclooctynyl group, so that it can perform a SPAAC reaction with the corresponding cyclooctyne group or azido group of the central core or the coupling arm, and then interact with the The core or the coupling arm is coupled. Or, in some embodiments, the second element carries a tetrazinyl or cyclooctenyl group, so that it can perform an iEDDA reaction with the corresponding cyclooctenyl or tetrazinyl group of the central core or the coupling arm, and further, The central core or the coupling arm is coupled. According to some embodiments, the engagement unit includes an engagement arm that is connected to the connection arm through a CuAAC or SPAAC reaction; in these cases, the N- or C-terminus of the central core or the free end of the coupling arm has four Azinyl or cyclooctenyl, so that the second element with the corresponding cyclooctenyl or tetrazinyl group can be connected to the central core or the coupling arm through the iEDDA reaction. According to other embodiments, the engagement unit includes an engagement arm that is connected to the connection arm through an iEDDA reaction; in these cases, the N- or C-terminus of the central core or the free end of the coupling arm has an azide group, Alkynyl or cyclooctynyl, so that the second element with the corresponding chemical group can be connected to the central core or the coupling arm through a CuAAC or SPAAC reaction.

In an optional embodiment of the present disclosure, when the first element is an effect element, the second element may be another effect element, which may be additively, synergistically, or independently of the first element. ) Effect; or, the second element may be a target element or an element capable of improving the pharmacokinetic properties of the junction unit, such as solubility, clearance, half-life, and bioavailability. In other optional embodiments, when the first element is a target element, the second element is preferably an effect element or an element capable of improving the pharmacokinetic properties of the junction unit.

In some embodiments, the bonding unit further includes a third element added as needed. Such a third element is different from the first and second elements. In the case where the second element is directly connected to the central core, the other end of the central core (that is, the free end not connected to the second element) may be a cysteine residue as required, and this residue may be used to Introduction of a non-essential third element. Specifically, the sulfhydryl group of a cysteine residue can react with the maleimide group on a PEG chain, and the PEG chain connected to the central core is referred to herein as a "coupling arm"; The free end of the coupling arm is provided with a tetrazinyl group or a cyclooctenyl group. In this way, the third element can be connected to the coupling arm through the iEDDA reaction. In a case where the bonding unit includes second and third elements, at least one of the first and second elements is preferably an effect element, and the third element may be an element capable of improving the pharmacokinetic properties of the bonding unit. A long PEG chain with a molecular weight of 20,000 to 50,000 daltons is an element that improves the pharmacokinetic properties of the junction unit.

＜ II ＞ Use of peptide-containing multi-arm conjugate

In the field of clinical medicine, the junction unit according to the first aspect of the present disclosure can be used for treating various diseases. Therefore, the second aspect of the present disclosure relates to methods for treating these diseases. According to various embodiments of the present disclosure, a method for treating a specific disease includes the steps of administering a therapeutically effective amount of an engaging unit to an individual in need, the engaging unit may be in accordance with the above aspects and embodiments of the present disclosure. Any type of joint unit. It will be understood that the junction unit may be administered in the form of a pharmaceutical formulation which, in addition to the junction unit of the present invention, also contains a pharmaceutically acceptable excipient suitable for the intended mode of administration.

In order that those skilled in the art to which this invention pertains can understand certain embodiments of the present disclosure, the following illustrates the combination of the first and second elements contained in the junction unit that can be used to treat certain specific diseases.

According to some embodiments of the present disclosure, the junction unit proposed herein can be used to treat central nervous system (CNS) diseases, such as multiple sclerosis and Alzheimer's disease. In the treatment of multiple sclerosis, the first element may be fingolimod, fingolimod phosphate, interferon-β or scFv specific to integrin-α4. When treating Alzheimer's disease, the first element may be a scFv specific to β-amyloid.

According to other embodiments of the present disclosure, the junction unit can be used to treat infectious diseases, and such junction unit comprises an scFv (as a first element) specific to a viral protein or a bacterial protein. In a preferred embodiment, the viral protein may be F protein of respiratory syncytial virus (RSV), gp120 protein of human immunodeficiency virus type 1 HIV-1, Hemagglutinin A (HA) protein of influenza A virus or glycoprotein of cytomegalovirus; bacterial protein can be Gram (-) bacteria Endotoxin, surface antigen of Clostridium difficile , lipoteichoic acid of Staphylococcus aureus , anthrax toxin of Bacillus anthracis , or Shiga-like toxin type I or II of Escherichia coli .

In order to make the description of this disclosure more detailed and complete, the following provides an illustrative description of the implementation mode and specific embodiments of the present invention; but this is not the only form of implementing or using the specific embodiments of the present invention. The embodiments include the features of a plurality of specific embodiments, as well as the method steps and their order for constructing and operating these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and sequence of steps.

For ease of explanation, some terms used in the specification, examples, and accompanying patent applications are compiled here. Unless otherwise defined in this specification, the meanings of scientific and technical terms used herein have the same meanings as understood and used by those having ordinary knowledge in the technical field to which the present invention belongs.

Without conflict with the context, the singular noun used in this specification covers the plural form of the noun; and the plural noun used also covers the singular form of the noun. In addition, in this specification and the scope of patent application, the expressions "at least one" and "one or more" have the same meaning, and both represent one, two, three, or more. What is more, in the scope of this specification and the patent application, "at least one of A, B, and C", "at least one of A, B, or C", and "at least one of A, B, and / or C" "Means covering only A, B, C, A, and B, B and C, and C, and A, B, and C.

Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate values, the relevant values in the specific embodiments have been presented here as accurately as possible. However, any numerical value inherently contains standard deviations due to individual test methods. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range. Alternatively, the word "about" means that the actual value falls within the acceptable standard error of the average value, depending on the consideration of those with ordinary knowledge in the technical field to which the present invention belongs. As can be understood, all ranges, quantities, values, and percentages used herein (such as to describe the amount of materials, length of time, temperature, operating conditions, quantity ratios, and others, except for experimental examples, or unless explicitly stated otherwise) Similar ones) are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the scope of the accompanying patent application are approximate values and may be changed as required. At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the value obtained by applying the general round method. Herein, the numerical range is expressed from one endpoint to the other endpoint or between two endpoints; unless otherwise stated, the numerical ranges described herein include the endpoints.

This disclosure generally relates to a variety of molecular constructs, where each molecular construct includes a target element (T) and an effector element (E). In this specification, these molecular constructs are sometimes referred to as "TE molecules", "TE Drug" or "TE Drug".

Here, the term "targeting element" refers to the part of a molecular construct that can directly or indirectly bind to a target (such as a receptor on a cell surface or a protein in a tissue), and thus can Assist in delivering the molecular construct of the invention to a target. In certain examples, the target element can direct the molecular construct to a nearby target cell. In other cases, the target element can specifically bind to a molecule on the surface of the target cell; or the target element can specifically bind to a second molecule, and this second molecule can specifically bind to a molecule on the surface of the target cell. In some examples, once the target element is engaged with the target target, the target element can internalize the molecule of the present invention and move it into the cytoplasm of the target cell. The target element can be an antibody or ligand of a cell surface receptor; or a molecule that can bind to the above antibody or ligand, and thus can indirectly target the molecular construct of the present invention to a target site (such as a selected cell surface). Compared with therapeutic drugs without target functions, the use of the molecular construct of the present invention can enhance or facilitate localization of effector elements (therapeutic agents) at the disease site. The above-mentioned localization is a degree or relative proportion, rather than an absolute or complete localization of the effector element at the disease site.

According to the present invention, the term "effector element" means that once the molecular construct has reached the target site, the molecular construct can exert biological activity (e.g., elicit an immune response, exert cytotoxicity, and the like). Or other functional activity (such as recruitment of other hapten-labeled therapeutic molecules). "Effect" may refer to a therapeutic or diagnostic effect. The effector element comprises an element capable of binding to a cell and / or an extracellular immunomodulatory factor. The above-mentioned effect elements include substances such as proteins, nucleic acids, lipids, carbohydrates, glycoproteins, pharmaceutical parts (including small molecule drugs and biological agents), compound elements, and isotopes, and also include fragments of the aforementioned substances.

Although the terms "first", "second," and "third" are used herein to describe various elements, parts, regions, and / or sections, these elements (and parts, regions, and / or sections) are not subject to the above Restrictions on modifiers. Furthermore, the use of these ordinal numbers does not imply a sequence or order, unless the context clearly indicates otherwise. Instead, these words are only used to distinguish the components. Therefore, the first element described below may also be named the second element without departing from what is disclosed in the exemplary embodiment.

Here, the terms "link", "couple" and "conjugate" are used interchangeably and are all used to refer to the connection of two components through a direct or indirect connection relationship Together.

The term "polypeptide" refers to a polymer having at least two amino acid residues. Generally, a polypeptide contains 2 to 200 amino acid residues; in the preferred case, 2 to 50 residues. The amino acid sequence mentioned in this specification covers the L-, D- or β-amino acid form of such a sequence. Polypeptides also include amino acid polymers, in which one or more amino acid residues are artificial chemical analogues corresponding to a natural amino acid, and of course also include naturally occurring amino acid polymers. In addition, this term also covers amino acids linked by polypeptide chains or other methods, such as modified linkage, such as α-ester, β-ester ), Thioamide, phosphoramide, carbomate, hydroxylate bonds, and the like to replace polypeptide bonds.

In some embodiments, the scope of the invention also encompasses other proteins that have conservative substitutions compared to the sequence. In various embodiments, one, two, three, four or five different residues are substituted. As used herein, the term "conservative substitution" means that the amino acid substitution does not significantly change the molecular activity (such as biological or functional activity and / or specificity). In general, conservative amino acid substitution is the replacement of an amino acid with another amino acid having similar chemical properties (such as charge or hydrophobicity). Some conservative substitutions include "analog substitutions", that is, the replacement of standard amino acids with non-standard (such as rare or synthetic) amino acids, and the above non-standard amino acids and substituted original amino acids The difference between them is minimal. The standard amino acid can be artificially modified to obtain the aforementioned amino acid analog, such as isomers or metabolic precursors, without greatly changing the original amino acid structure.

In some embodiments, the scope of the present invention also covers any sequence that has at least 80% sequence similarity to any of the described sequences. The sequence similarity is preferably at least 85% or 90%, more preferably at least 95% or 98%. .

% 其中X是利用BLAST序列並排程式對序列A、B進行排列後所得到的相同胺基酸殘基數目(identical matches)，而Y是A、B二序列中較短者的胺基酸殘基總數。The `` Percentage (%) amino acid sequence identity '' described herein for a polypeptide sequence means that the amino acid residue of the candidate sequence is identical to the amino acid residue of the reference polypeptide sequence. Percentage; when performing the above-mentioned alignment, the candidate polypeptide fragment and the specific polypeptide fragment may be side by side, and a gap may be introduced when necessary, so that the two sequences form the highest sequence similarity; when calculating the similarity, Conservatively substituted amino acid residues are considered different residues. Various methods have been used in the related art to perform the above-mentioned side-by-side, such as publicly available software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). Those with ordinary knowledge in the technical field to which the present invention belongs may select appropriate parameters and calculation methods when performing side-by-side to obtain the optimal arrangement. In this specification, the sequence comparison between the two polypeptide sequences is performed using the protein-protein BLAST analysis database Blastp provided by the National Center for Biotechnology Information (NCBI). Calculation of the amino acid sequence similarity of the candidate polypeptide sequence A compared to the reference polypeptide sequence B (also referred to in this specification as the similarity of the amino acid sequence of the polypeptide sequence A and the polypeptide sequence B with a specific percentage (%)) Here's how:

% Where X is the number of identical amino acid residues (identical matches) obtained by aligning sequences A and B using the BLAST sequence side-by-side program, and Y is the shorter amino acid residue in the A and B sequences total.

The term "PEGylated amino acid" refers herein to a polyethylene glycol (PEG) chain with an amino group and a carboxyl group. Generally, the structural formula of pegylated amino acid is NH _2- (CH ₂ CH ₂ O) _n -COOH. In this disclosure, the value of n is between 1 and 20; preferably between 2 and 12.

The "terminal" of a polypeptide herein refers to an amino acid residue at the N- or C-terminus of the polypeptide. When describing the constituent units of a polymer, such as polyethylene glycol described herein, "end" refers to the portion at the end of the polymer backbone. In this specification and the scope of the patent application, the term "free end" is used to refer to a terminal amino acid residue or a constituent unit that does not form a chemical bond with another molecule.

The term "antigen or Ag" refers to a molecule capable of causing an immune response. Such immune responses may involve secretory, humoral, and / or cellular antigen-specific responses. In the present disclosure, the term "antigen" refers to a protein, a polypeptide (including a mutant strain or a biologically active fragment thereof), a polysaccharide, a glycoprotein, a glycolipid, a nucleic acid, or a combination thereof.

In the scope of this description and the patent application, the term "antibody" should be interpreted in a broad sense and includes a fully assembled antibody, an antibody-binding antibody fragment, such as an antigen-binding fragment (Fab / Fab '), F (ab ') ₂ fragments (having two Fab portions connected to each other by a double sulfur bond), variable fragments (Fv), single-stranded variable fragments (scFv), bispecific single-stranded variable fragments (bi -scFv), nanobodies, unibodies, and diabodies. An "antibody fragment" comprises a portion of an intact antibody, and preferably includes an antigen-binding region or a variable region of the intact antibody. In a typical example, "antibody" refers to a protein consisting essentially of one or more polypeptides encoded by an immunoglobulin gene or a fragment thereof. Conventional immunoglobulin genes include constant region genes such as κ (kappa), λ (lambda), α (alpha), γ (gamma), δ (gamma), ε (epsilon), and μ (mu). ), And countless immunoglobulin variable region genes. Light chains are usually classified as either kappa or lambda. Heavy chains are generally classified as γ (gamma), μ (mu), α (alpha), δ (gamma), or ε (epsilon). Based on these structures, the following types of immunoglobulins are defined: IgG, IgM, IgA , IgD, and IgE. A typical immunoglobulin antibody structure is a tetramer. Each tetramer is composed of two identical polypeptide chains, and each pair has a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region containing about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The terms variable light chain (V _L ) and variable heavu chain (V _H ) refer to the above light chain and heavy chain, respectively. According to an embodiment of the present disclosure, the above antibody fragments can be resynthesized by changing a natural antibody or using a recombinant DNA method. In some embodiments of the present disclosure, the above-mentioned antibodies and / or antibody fragments may have dual specificity, and may have multiple different configurations. For example, a bispecific antibody may include two different antigen binding sites (variable regions). In various embodiments, bispecific antibodies can be prepared using fusion tumor technology or recombinant DNA technology. In some embodiments, a bispecific antibody has binding specificity for at least two different epitopes.

The term "specifically bind" refers to the ability of an antibody or an antigen-binding fragment thereof to bind to an antigen. The dissociation constant (Kd) of the above binding is less than about 1 × 10 ^{− 6} M, 1 × 10 ^{− 7} M, 1 × 10 ^{− 8} M, 1 × 10 ^{− 9} M, 1 × 10 ^{− 10} M, 1 × 10 ^{− 11} M, or 1 × 10 ^{− 12} M; or compared to or in addition to It has a binding affinity for non-specific antigens, and the affinity of the above-mentioned antibody or antigen-binding fragment thereof to the antigen is more than twice.

The term "treatment" refers to prophylactic (e.g., prophylactic), healing or palliative treatments to achieve the desired pharmaceutical and / or physiological effect; and the act of treatment includes the prevention described above Sexual, healing or palliative treatment. Specifically, treatment herein refers to administering or administering a molecular construct of the present invention or a medicament containing the molecular construct to an individual who may have a medical condition, symptom, disease, or disorder associated with the condition, or is susceptible to the condition. A composition that is capable of partially or completely alleviating, ameliorating, alleviating one or more symptoms or characteristics of a particular abnormality and / or condition, or delaying its occurrence, hindering its progress, reducing its severity, and / or reducing its incidence. Treatment may also be performed on individuals who have not shown signs of a disease, disorder, and / or disorder, and / or individuals who show early signs, with a view to reducing the risk of developing pathological changes associated with the disease, disorder, and / or disorder.

As used herein, the term "effective amount" means that the molecular construct of the present invention is used in an amount sufficient to induce the desired therapeutic response. An effective amount of an agent is not necessarily capable of curing a disease or disorder, but it can delay, hinder or prevent the occurrence of the disease or disorder, or it can reduce the symptoms associated with the disease or disorder. A therapeutically effective amount can be divided into one, two or more doses and administered once, two or more times in a suitable dosage form over a specified period. The specific therapeutically effective amount depends on a variety of factors, such as the particular condition being treated, the individual's physiological conditions (e.g., individual weight, age, or gender), the type of individual being treated, the duration of the treatment, and concurrent treatment (if any) The nature of the compound and the specific formula used and the structure of the compound or its derivative. For example, a therapeutically effective amount can be expressed as the total weight of the active ingredient, such as grams, milligrams, or micrograms; or as the ratio of the weight of the active ingredient to body weight, such as milligrams per kilogram of body weight (mg / mg / kg).

The terms "application" and "administration" are used interchangeably herein and refer to providing a molecular construct or a pharmaceutical composition of the present invention to an individual in need of treatment.

The terms "subject" or "patient" are used interchangeably herein and refer to animals (including humans) that can be treated with the molecular constructs, pharmaceutical compositions, and / or methods of the present disclosure. Unless otherwise specified, "individuals" or "patients" generally include males and females. "Individual" or "patient" includes any mammal that would benefit from the disposal of this disclosure. Examples of the "individual" or "patient" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and birds. In one example, the patient is a human. The mammals include all members of the mammalian class, including humans, primates, domestic and agricultural animals (such as rabbits, pigs, sheep, and cattle), zoo animals or race animals, pets, and rodents (such as, Mouse and rat). The term "non-human mammal" covers all members of the mammalian class except humans.

This disclosure is based at least in part on the construction of T-E drugs, which can be delivered to target cells, target tissues, or organs, and their proportions in these parts are relatively higher than in the blood circulation, lymphatic system, and other cells , Tissues and organs. When the above situation is achieved, the efficacy of T-E drugs can be improved, and the number and severity of side effects and toxicity will be reduced. Compared to drugs without target components, when the drug is delivered in the form of T-E molecules, the concentration of the effector that exerts the effect may be lower. Therefore, therapeutic effectors can be administered at lower doses without compromising their effectiveness, but at the same time reducing their side effects and toxicity.

Diseases that could benefit from better drug targets

If the drug can be targeted to the disease site, that is, if the drug can be localized at the disease site (compared to normal tissues or organs) or has a superior concentration, the drug used to treat many diseases can be improved and made available. Better efficacy and safety. Some antibody drugs are targeted at infectious microorganisms or their toxin products. If these antibody drugs have the ability to recruit immune cells to swallow or clear particles bound to antibodies, they can improve their efficacy. The following are some of the main exemplary diseases that can be improved if the drugs used in these diseases can have a better distribution ratio at the disease site or cells or can recruit phagocytic immune cells.

I Central nervous system disease

When treating central nervous system (CNS) diseases, it is usually necessary to pass the therapeutic agent through a blood-brain barrier (BBB) to enter the central nervous system. Some therapeutic agents do not enter the central nervous system; such drugs regulate some conditions in the central nervous system by regulating certain peripheral activities (such as immune activity). BBB is formed by endothelial cells that line the microvessels in the CNS. Unlike microvessels in surrounding tissues and organs, microvascular endothelial cells in the BBB are connected by tight junctions formed by occludin, claudin, and junctional adhesion molecules. Together.

β-amyloid is one of the main causes of Alzheimer's disease. At present, at least six antibodies specific to β-amyloid have been developed in clinical applications. They are: aducanumab, Bapineuzumab, crenezumab, gantenerumab, ponezumab, and solanizumab. The effectiveness of these antibodies in improving Alzheimer's disease is often suboptimal. This is believed to be because for these antibodies to be effective, a very large amount of antibodies must pass through the BBB to enter the damaged site in the CNS. However, only very small amounts of antibodies can pass the BBB.

Interferon-β-1a (IFN-β-1a) and interferon-β-1b (IFN-β-1b) are known to treat multiple sclerosis (MS). IFN-β-1a is produced by mammalian cells, while IFN-β-1b is produced by E. coli; these drugs are single-chain proteins consisting of 166 amino acid residues with a disulfide key. The above-mentioned therapeutic agents are said to reduce the MS relapse rate in treated patients to 18-38%. The mechanism of action of IFN-β-1a and IFN-β-1b is very complex and not completely understood by researchers. The process involves the positive regulation of anti-inflammatory immune cells and factors and the negative direction of pro-inflammatory T cells and factors. Regulation. Administration of IFN-β to MS patients also reduces the number of pro-inflammatory T cells that pass through the BBB. However, it is still unclear whether IFN-β-1a and IFN-β-1b exert their effects at least in part by entering the damaged site in the CNS.

When antibodies or protein drugs are applied to the body's peripheral system, only a small portion (about 0.1%) will reach the CNS because only a small portion of protein drugs can penetrate the BBB. However, it is known that in many CNS diseases (including Alzheimer's disease and multiple sclerosis), the inflammatory response at the affected site will cause the BBB defense line to disintegrate, thereby increasing the permeability of the site. Therefore, the present invention believes that if a relatively large amount of anti-β-amyloid or IFN-β-1a and IFN-β-1b antibodies can be directed to the BBB, the therapeutic agent has a higher chance to improve the efficacy through the BBB.

What's more, some therapeutic agents have been developed to inhibit the passage of inflammatory immune cells through the BBB. One of the most famous examples is natalizumab, which is specific for the cell adhesion molecule integrin α4. The antibody can inhibit the attachment of inflammatory immune cells to the BBB lining the endothelial layer and penetrate the BBB, thereby exerting its function. Although natalizumab has been shown to be effective, this drug has severe immunosuppressive side effects. Specifically, natalizumab causes progressive multifocal leukoencephalopathy, an opportunistic infection caused by the JC virus (John Cunningham virus). Therefore, the present invention believes that if a large amount of anti-integrin α4 antibody can be recruited to the BBB, a better therapeutic effect can be achieved at a lower dosage, and thus side effects can be reduced.

The endothelial cells in the microvessels that form BBB can express transferrin receptors and insulin receptors. These receptors can respectively regulate the transference of transferrin and insulin molecules to the cerebral parenchyma. ). When a transferrin receptor is used as a transport channel, only a small portion can penetrate and the vast majority of the remaining is trapped or degraded. Because endothelial cells lined with microvessels in other parts of the vasculature do not show transferrin receptors, transferrin receptors on endothelial cells in BBB can serve as site-specific antigens and can be used to recruit Therapeutics. Once the therapeutic agent is concentrated in the BBB, a higher proportion of the therapeutic agent can penetrate the microvessels.

The present invention also recognizes that once the mechanism for directing drugs to the BBB is established, the efficacy of various anti-inflammatory drugs to treat multiple CNS diseases can be further explored; the above drugs such as anti-TNF-α, anti-IL12 / IL-23 Anti-IL17 and anti-CD3.

For antibody drugs specific to integrin α4, transferrin receptors can be used as target site recruiters. In the treatment of Alzheimer's disease, the effector can be several scFvs specific to β-amyloid; in the treatment of multiple sclerosis, the effector can be several IFN-β-1a or IFN-β-1b Or several scFvs specific for integrin α4.

Various embodiments of the present disclosure disclose several TE molecules, which may be a single multi-armed junction configuration or a joint-linker configuration, each with a specificity for the transferrin receptor ScFv (as a target element) and scFv (as an effector element) that is specific for IFN-β-1a or IFN-β-1b or for integrin α-4. Alternative embodiments disclose a variety of TE molecules, which can be a single multi-armed conjugate configuration or a joint conjugate configuration, each with a scFv (as a target element) specific to the transferrin receptor, and ScFv (as an effector element) specific to β-amyloid.

Fingolimod is an immunosuppressive drug, a derivative of myriocin, a natural product isolated from certain fungi. Fingolimod has been licensed to reduce the recurrence of relapsing-remitting multiple sclerosis. In vivo, fingolimod is phosphorylated to form fingolimod phosphate, which is similar in structure to naturally occurring sphingosine-1-phosphate (S1P); fingolimod Tetraphosphate is an extracellular lipid medium and can be combined with four of the five S1P receptors. S1P receptors appear on lymphocytes and are involved in lymphocyte migration. One of the common pharmacological mechanisms of fingolimod is the inhibition of lymphocytes leaving the lymphoid tissue and entering the circulation (and therefore the CNS). Fingolimod can pass through the BBB and enter the CNS, and many types of cells in the CNS will display S1P receptors, which are related to cell expansion, shape and migration. Fingolimod is believed to work directly on the CNS. Common side effects from fingolimod administration include headaches and fatigue; they can also cause severe side effects such as skin cancer, macular edema, and fatal infections (such as hemorrhagic local encephalitis).

A fingolimod molecule has an NH ₂ group, which can be coupled through this functional group with a dual-use binding arm having an NHS group. According to a preferred embodiment of the present disclosure, a TE construct can be prepared with a standard element to deliver the construct to the BBB, and a fingolimod drug-loaded bundle as an effector element. For fingolimod drug-loaded bundles, 5 to 10 fingolimod molecules can be added to a binding unit; for example, fingolid can be cleavable linker or non-cleavable linker The mode molecule is coupled to the connecting arm of the junction unit. Because fingolimod phosphate is converted into the active form of fingolimod phosphate (similar to S1P) by patients, fingolimod phosphate can also be used to prepare drug-loaded bundles. The junction unit with fingolimod or fingolimod phosphate-loaded bundles can be coupled to one or two scFvs specific to transferrin receptor I. After the molecular construct is administered, a portion of the molecular construct is brought to the BBB. Fingolimod molecules released from the cleavable binding arm can pass the BBB and enter the CNS. Alternatively, there are partially complete constructs that can enter the CNS. A variety of cleavage mechanisms can be used to design cleavable engagement arms. A common method is to add the SS bond, and the SS disulfide bond can be cleaved by a reduction reaction at the target tissue site. Another common cleavable bond in the field of conjugate design is the use of polypeptide bonds between amino acids that are sensitive to proteases such as matrix metalloproteinases.

Some exemplary TE molecules can be a single multi-armed conjugate configuration or a combined conjugate configuration, each with one or two scFvs (as the target element) specific to the transferrin receptor. De (as an effect element). In all such molecular constructs, the connection between the connecting arm and the effector element can be an uncleavable or a cleavable bond. When the molecular construct proposed by the present invention is used to treat various CNS diseases, the target portion may have one or two scFvs specific to a transferrin receptor or an insulin receptor. One or two scFvs specific to the transferrin receptor or insulin receptor can be used. If the scFv used has a relatively high affinity (Kd <1x10 ^-9 ), one scFv can be used; if the scFv used has a general affinity (1x10 ^-9 <Kd <5x10 ^-8 ), two scFvs can be used. In a better case, more than two scFvs specific to the transferrin receptor or insulin receptor will not be used to avoid receptor cross-linking and endocytosis of the bound drug.

II Infectious disease

Although a large number of monoclonal antibodies have been prepared against a variety of viruses, bacteria, and fungi that can cause severe infections in humans or animals, only a few monoclonal antibodies are licensed as preventive or therapeutic drugs to fight infection. This deficiency can be attributed to several major factors, the most important of which is that these infectious microorganisms and their products have many different serotypes and have different degrees of response to specific antibodies. Another reason is that the target microorganisms are mutated, thus avoiding the response caused by specific antibodies.

The T-E molecular design proposed by the present invention can also be used to prevent and treat infectious diseases. The plurality of linking arms can improve the binding affinity and specificity of the target infectious microorganism or its product, and can induce immune function to clear the microorganism and its product. The present invention believes that improving binding affinity and inducing immune clearance function can overcome the problems of serotype differences and mutations to a certain extent. Such improvements can enhance the effectiveness of candidate antibodies in preventing and treating infectious diseases. Many antibodies that fail to achieve the desired effect in clinical trials can be redesigned according to the present invention and tested again for their effects.

A preferred set of embodiments of the present invention uses a joint conjugate configuration with a junction unit for the target and a junction unit for recruiting effector functions. Another preferred embodiment uses a single joint unit having a plurality of connecting arms for connecting the target element and a coupling arm for connecting the effect element. The target element can be any of the following two categories: (1) scFv or sdAb specific to the surface components of microorganisms or products of microorganisms, such as the membrane protein gp120 of human immunodeficiency virus type I (HIV-1), the respiratory tract Fusion virus (RSV) F protein, surface antigen of Clostridium difficile or Staphylococcus aureus, or endotoxin of gram-negative bacteria or Shiga toxoid of E. coli; or (2) a cell surface receptor of the virus Extracellular parts, such as the gp120-binding CD4 domain of HIV-1.

The effector element can be one or two scFv or sdAb specific to the Fc receptor of IgG; the above receptors are, for example, FcγRIIA (CD32), FcγRIIIB (CD16b), or FcγRI (CD64). These receptors appear on neutrophils, macrophages, and eosinophils, and are the main molecules that regulate the phagocytosis of antibody-bound microorganisms. FcγRIIA and FcγRIIIB can bind to IgG, but have lower affinity (Kd is about ^10-6 to ^10-7 ); while FcγRI can bind to IgG1 and IgG3, and have higher affinity (Kd about ^10-9 ). In the best case, scFv or sdAb specific to FcyRIIA or FcyRIIIB can be used because these molecules have advantages when competing with IgG for receptor binding.

Antibodies that are specific for carbohydrate antigens on the surface of bacteria usually have poor affinity and are mainly expressed on IgM and not on IgG. IgM molecules have 10 Fv (antigen-binding sites). However, the molecular weight of an IgM molecule is about 1,000 kDa, which cannot pass through microvessels to reach extravascular space. When using the configuration proposed by the present invention, the molecular construct with six scFv or 10 sdAb has a molecular weight of about 150 kDa.

When using antibody drugs to destroy the virus, one of the keys is that the drug cannot trigger FcR to promote viral infection. In such cases, the combined virions are not swallowed and digested. Some viruses (such as dengue virus) expand in phagocytic cells. Therefore, if the virus particles can approach the cell and enter the bound cell without being destroyed, the virus can expand within the infected cell. Therefore, according to a preferred set of embodiments of the present invention, the molecular construct is provided with two or more scFvs specific to the Fcγ receptor, which can bind to multiple Fcγ receptor molecules on the surface of phagocytic cells, so that The bound virus molecules must enter the phagocytosis pathway.

A variety of antibodies specific to viruses, bacteria or their products have entered clinical trials, but only one antibody specific to RSV has been approved for marketing. Even with the aforementioned RSV-specific antibodies, the approved uses are limited to prevention and cannot be used to treat ongoing infections. There is an urgent need in the art to design anti-RSV antibodies that can be used to treat already infected individuals. Other antibodies are still in clinical development or fail clinical trials. Using the molecular construct platform proposed by the present invention, these antibodies can be used and their efficacy improved. The antibodies include at least the following antibodies: (1) palivizumab and felvizumab specific to the F protein of the respiratory fusion virus; (2) specific to human immunodeficiency virus type I gp120 Suweizumab; (3) Livivirum, exbivirumab, and tuvirumab specific to hepatitis B virus hepatitis B surface antigen (HBsAg) ); (4) CR6261 monoclonal antibody specific to influenza A virus hemagglutinin A and firvumab; (5) regavirumab and sevevir specific to cytomegalovirus glycoprotein Monoclonal antibody (sevirumab); (6) rafivirumab specific to rabies virus glycoprotein; (7) actoxumab and betexurin specific to Clostridium difficile surface antigen MAb (bezlotoxumab); (8) Obitoxaximab and raxibacumab specific to anthrax anthracnose; (9) Specific to serotype IATS O11 of Pseudomonas aeruginosa Panobacumab (panobacumab; a human IgM monoclonal antibody); (10) golden Tefibazumab and tosatoxumab specific to staphylococcus agglutination factor A; (11) edobacomab specific to Gram-negative endotoxin; (For the treatment of sepsis); (12) pagibaximab specific for staphylococcal wallic acid; (13) raxibacumab (raxibacumab; human) specific for anthrax toxin Monoclonal antibodies); (14) Prituxaximab, setoxaximab, and urtoxazumab specific to E. coli type I or type II shiga toxins.

According to several embodiments of the present disclosure, TE molecules used to treat infectious diseases may adopt a joint conjugate configuration that includes F protein for respiratory fusion virus (RSV) or human immunodeficiency virus type I (HIV- 1) The scpv specific to gp120 is used as the target / capture element, and the scFv specific to FcγRIIA (CD32) or FcγRIIIB (CD16b) is used as the effect / clearance element. According to an embodiment of the present disclosure, certain TE molecules may adopt a single junction unit or a joint junction configuration that includes scFv specific to Gram-negative endotoxin or Staphylococcus aureus as the target / capture element , And scFv specific to CD32 or CD16b as the effect / clearing element. In the process of Gram-negative bacteria infection, for the potentially life-threatening symptoms of sepsis, the accelerated removal of endotoxin should reduce the large release of interleukins (such as TNF-α, IL-1, etc.) (i.e. , Interleukin Storm). When the molecular construct platform of the present invention is administered to treat an infectious disease, the effector part may carry one or two scFvs specific to CD32 or CD16b. One or two scFvs specific to CD32 (CD32a or CD32b) or CD16b can be used. If the scFv used has a relatively high affinity (Kd <1x10 ^-9 ), one scFv can be used; if the scFv used has a general affinity (1x10 ^-9 <Kd <5x10 ^-8 ), two scFvs can be used. In a better case, more than two scFvs specific to CD32 or CD16b will not be used to avoid receptor cross-linking and endocytosis of the bound drug.

Section One Multi-arm conjugate for treating specific diseases

(i) Polypeptide core for multiarm conjugates

A first aspect of the present disclosure relates to a junction unit comprising: (1) a central core comprising 2-15 lysine (K) residues; and (2) 2-15 linking arms, Each K residue is linked to a central core, respectively. The central core of the present invention is characterized in that its N- or C-terminus bears an azide group, an alkynyl group, a tetrazinyl group or a high-stress alkynyl group.

In preparing the bonding unit of the present invention, one end bears an N-hydroxysuccinimydil (NHS) group and the other end bears a functional group (for example, NHS group, maleimide diimide group, Nitrogen, alkynyl, tetrazinyl, or high-stress alkynyl) PEG chains are attached to the K residue; specifically, the above NHS group and the amino group on the K residue are used to form an amidine bond, which in turn connects the PEG chain Go to the core. In the present disclosure, the PEG chain attached to the K residue is referred to as a linking arm, and the free end of this linking arm carries a functional group.

According to an embodiment of the present disclosure, the above-mentioned central core is a polypeptide core, which is 8-120 amino acid residues in length and contains 2 to 15 lysine (K) residues, where each K residue and The next K residues are separated by a padding sequence.

According to an embodiment of the present disclosure, the stuffing sequence comprises glycine (G) and serine (S) residues; in a preferred case, the stuffing sequence is selected from the group consisting of G, S, and Composed of 2-15 residues. For example, the padding sequence can be: GS, GGS, GSG, GGGS (sequence number: 1), GSGS (sequence number: 2), GGSG (sequence number: 3), GSGGS (sequence number: 4), SGGSG (sequence Number: 5), GGGGS (Serial Number: 6), GGSGGS (Serial Number: 7), GGSGGSG (Serial Number: 8), SGSGGSGS (Serial Number: 9), GSGGSGSGS (Serial Number: 10), SGGSGGSGSG (Serial Number: 11), GGSGGSGGSGS (serial number: 12), SGGSGGSGSGGS (serial number: 13), GGGGSGGSGGGGS (serial number: 14), GGGSGSGSGSGGGS (serial number: 15), or SGSGGGGGSGGSGSG (serial number: 16).

The stuffing sequence between two lysine residues may consist of glycine and serine residues of various lengths and combinations. In polypeptide cores with a small number of lysine residues, longer filler sequences can be used; in polypeptide cores with a large number of lysine residues, shorter filler sequences can be used. In addition to glycine and serine, hydrophilic amino acid residues such as aspartic acid and histidine can be added to the filling sequence. In addition to the stuffing sequence consisting of glycine and serine residues, other stuffing sequences can also be used. For example, flexible and soluble loops in common human serum proteins such as albumin and immunoglobulin can be used.

According to some preferred embodiments of the present disclosure, the central core contains a G _1-5 SK sequence of 2-15 units. Alternatively, the sequence of this polypeptide core is (GSK) _2-15 ; that is, the polypeptide comprises at least two consecutive GSK units. For example, the central core of the present invention may include the following amino acid sequences: Ac-CGGSGGSGGSKGSGSK (sequence number: 17), Ac-CGGSGGSGGSKGSGSKGSK (sequence number: 18) or Ac-CGSKGSKGSKGSKGSKGSKGSKGSKGSKGSK (sequence number: 19), where Ac represents B醯 基.

According to certain embodiments of the present disclosure, the central core is a polypeptide core having the sequence (X _aa -K) _n , where X _aa is a pegylated amino acid having 2 to 12 ethylene glycol (EG) repeats Unit, and n is an integer from 2 to 15.

As described above, the central core of the present invention is characterized in that its N- or C-terminus bears an azide group, an alkynyl group, a tetrazinyl group, or a high-stress alkynyl group. According to certain embodiments of the present disclosure, the N- or C-terminal amino acid residue of the central core of the present invention carries an azide or alkynyl group. Amino acid residues bearing azido groups may be: L-azidohomogenine (AHA), 4-azido-L-phenylalanine, 4-azido-D-phenylalanine, 3-azido- L-Alanine, 3-Azide-D-Alanine, 4-Azide-L-Homoalanine, 4-Azide-D-Homoalanine, 5-Azide-L-Uranine, 5- Azide-D-uramine, 6-azide-L-lysine or 6-azide-D-lysine. For example, the central core of the present invention may have the following sequences: Ac- (GSK) _2-7- (G _2-4 S) _1-8 -A ^AH , Ac-A ^AH- (SG _2-4 ) _1-8 -(GSK) _2-7 , Ac-A ^AH- (SG _2-4 ) _0-7- (GSK) _2-6- (G _2-4 S) _1-8 -C, Ac-C- (SG _{2 -4} ) _0-7- (GSK) _2-6- (G _2-4 S) _1-8 -A ^AH , Ac-K- (X _{aa 2-12} -K) _2-4 -X _{aa 2-12} ^{-A AH, Ac-A AH -X} aa 2-12 -K- (X aa 2-12 -K) 2-4, Ac-A AH -X aa 2-12 -K- (X aa 2-12 - K) _1-3 -X _{aa 2-12} -C or Ac-CX _{aa 2-12} -K- (X _{aa 2-12} -K) _1-3 -X _{aa 2-12} -A ^AH , where X _aa is A PEGylated amino acid with the stated number of EG repeat units, Ac represents ethenyl, and A ^AH represents AHA residues.

Examples of amino acids having an alkynyl group include, but are not limited to: L-high propargyl glycine (L-HPG), D-high propargyl glycine (D-HPG), or β-high Propargyl Glycine (β-HPG). In this example, the central core of the present invention may have the following sequences: Ac- (GSK) _2-7- (G _2-4 S) _1-8 -G ^HP , Ac-G ^HP- (SG _2-4 ) _{1- 8-} (GSK) _2-7 , Ac-G ^HP- (SG _2-4 ) _0-7- (GSK) _2-6- (G _2-4 S) _1-8 -C, Ac-C- (SG _2-4 ) _0-7- (GSK) _2-6- (G _2-4 S) _1-8 -G ^HP , Ac-K- (X _{aa 2-12} -K) _2-4 -X _{aa 2- 12} -G ^HP , Ac-G ^HP -X _{aa 2-12} -K- (X _{aa 2-12} -K) _2-4 , Ac-G ^HP -X _{aa 2-12} -K- (X _{aa 2-12} -K) _1-3 -X _{aa 2-12} -C or Ac-CX _{aa 2-12} -K- (X _{aa 2-12} -K) _1-3 -X _{aa 2-12} -G ^HP , where X _aa Is a pegylated amino acid with the stated number of EG repeat units, Ac stands for ethenyl, and G ^HP stands for HPG residue.

As can be understood, a variety of amino acids and pegylated amino acids with azide or alkynyl groups on the side chains are currently available through commercial channels. These amino acids may have protective groups such as t-butyl Tert-butyloxycarbonyl (t-boc) or 9-fluorenylmethyloxycarbonyl (Fmoc) can be easily used for solid peptide synthesis.

According to some experimental examples of this disclosure, the central core used may include the following sequences: Ac-G ^HP GGSGGSGGSKGSGSK (serial number: 21), Ac-G ^HP GGSGGSGGSKGSGSKGSK (serial number: 22), Ac-A ^AH GGSGGSGGSKGSGSKGSK (serial number : 23), Ac-G ^HP GGSGGSGGSKGSGSKGSGSC (serial number: 24), Ac-CX _{aa 2} -KX _{aa 2} -KX _{aa 2} -K (serial number: 25) or Ac-CX _{aa 6} -KX _{aa 6} -KX _{aa 6} -KX _{aa 6} -KX _{aa 6} -K (sequence number: 26), where X _aa is a pegylated amino acid with a specified number of EG repeat units, Ac represents ethenyl, and A ^AH represents AHA residue And G ^HP stands for HPG residue.

Alternatively, the central core of the present invention may be connected to a coupling arm, and the free end of the coupling arm (ie, the end not connected to the central core) carries a functional group (such as azide, alkynyl, tetrazinyl, or high stress). Alkynyl). In these cases, the N- or C-terminus of the core of the invention is a cysteine residue. When preparing a junction unit connected to a coupling arm, a PEG chain having a cis-butene diimide group at one end and the above-mentioned functional group at the other end is connected to the above cysteine residue; specifically, it can be transmitted through The sulfhydryl-maleimide reaction between the sulfhydryl group of the PEG chain and the sulfhydryl group of the cysteine residue links the two together. In the present disclosure, the PEG chain attached to the cysteine residue at the central core terminal is called a coupling arm, and the free end thereof bears the above-mentioned functional group.

In the preferred case, the free end of the coupling arm bears a tetrazinyl or high-stress alkynyl (eg, cyclooctenyl or cyclooctynyl). These coupling arms have 2-12 EG units. According to an embodiment of the present disclosure, the tetrazinyl group is 1,2,3,4-tetrazine, 1,2,3,5-tetrazine 1,2,4,5-tetrazine or a derivative thereof. The high-stress alkynyl may be cyclooctenyl or cyclooctynyl. According to the experimental examples of the present disclosure, the cyclooctenyl group may be a trans-cyclooctene (TCO) group; and examples of the cyclooctynyl group include, but are not limited to, dibenzocyclooctyne (DBCO) , Difluorinated cyclooctyne (DIFO), dicyclononyne (BCN) and dibenzocyclooctyne (DICO). According to some embodiments of the present disclosure, the tetrazinyl is 6-methyl-tetrazine.

According to various embodiments, central core for the arm connected to the coupling include, but are not limited _{to: Ac- (GSK) 2-7 - (} G 2-4 S) 1-8 -CX aa 2-12 - tetrazine, Ac _{- (GSK) 2-7 - (G} 2-4 S) 1-8 -CX aa 2-12 - high stress _{alkynyl, Ac-K- (X aa 2-12} -K) 2-4 -X aa 2 _-12 -CX _{aa 2-12} -tetrazine, Ac-K- (X _{aa 2-12} -K) _2-4 -X _{aa 2-12} -CX _{aa 2-12} -high stress alkynyl, tetrazinyl- X _{aa 2-12} -C (Ac)-(SG _2-4 ) _1-8- (GSK) _2-7 , High stress alkynyl-X _{aa 2-12} -C (Ac)-(SG _2-4 ) _1-8 - (GSK) _2-7, tetrazine group _{-X aa 2-12 -C (Ac) -X} aa 2-12 -K- (X aa 2-12 -K) 2-4 alkynyl, and high stress The group -X _{aa 2-12} -C (Ac) -X _{aa 2-12} -K- (X _{aa 2-12} -K) _2-4 .

Alternatively, one end of the central core is provided with an azide group or an alkynyl group, and the other end is connected with a coupling arm having a tetrazinyl group or a high-stress alkynyl group. Here is an example: Ac-A ^AH- (SG _{2- 4) 0-7 - (GSK) 2-6} - (G 2-4 S) 1-8 -CX aa 2-12 - ^{tetrazine, Ac-A AH - (SG} 2-4) 0-7 - (GSK _{) 2-6 - (G 2-4 S)} 1-8 -CX aa 2-12 - high stress alkynyl group, tetrazine group _{-X aa 2-12 -C (Ac) -} (SG 2-4) 0- _7- (GSK) _2-6- (G _2-4 S) _1-8 -A ^AH , high stress alkynyl-X _{aa 2-12} -C (Ac)-(SG _2-4 ) _0-7- ( _{GSK) 2-6 - (G 2-4 S} ) 1-8 -A AH, Ac-A AH -X aa 2-12 -K- (X aa 2-12 -K) 1-3 -X aa 2- ₁₂ -CX _{aa 2-12} -tetrazine, Ac-A ^AH -X _{aa 2-12} -K- (X _{aa 2-12} -K) _1-3 -X _{aa 2-12} -CX _{aa 2-12} -high Stress alkynyl, tetrazinyl-X _{aa 2-12} -C (Ac) -X _{aa 2-12} -K- (X _{aa 2-12} -K) _1-3 -X _{aa 2-12} -A ^AH , high Stress alkynyl-X _{aa 2-12} -C (Ac) -X _{aa 2-12} -K- (X _{aa 2-12} -K) _1-3 -X _{aa 2-12} -A ^AH , Ac-G ^HP- _{(SG 2-4) 0-7 - (GSK} ) 2-6 - (G 2-4 S) 1-8 -CX aa 2-12 - ^{tetrazine, Ac-G HP - (SG} 2-4) 0- _{7 - (GSK) 2-6 - (} G 2-4 S) 1-8 -CX aa 2-12 - high stress alkynyl group, tetrazine group _{-X aa 2-12 -C (Ac) -} (SG 2- ₄ ) _0-7- (GSK) _2-6- (G _2-4 S ) _1-8 -G ^HP , high stress alkynyl-X _{aa 2-12} -C (AC)-(SG _2-4 ) _0-7- (GSK) _2-6- (G _2-4 S) _{1- 8} -G ^HP , Ac-G ^HP -X _{aa 2-12} -K- (X _{aa 2-12} -K) _1-3 -X _{aa 2-12} -CX _{aa 2-12} -tetrazine, Ac-G ^HP -X _{aa 2-12} -K- (X _{aa 2-12} -K) _1-3 -X _{aa 2-12} -CX _{aa 2-12} -high stress alkynyl, tetrazinyl-X _{aa 2-12} -C (Ac) -X _{aa 2-12} -K- (X _{aa 2-12} -K) _1-3 -X _{aa 2-12} -G ^HP and high stress alkynyl-X _{aa 2-12} -C (Ac)- X _{aa 2-12} -K- (X _{aa 2-12} -K) _1-3 -X _{aa 2-12} -G ^HP .

Recombinant technology can also be used to synthesize the above-mentioned polypeptides, and then use bacterial or mammalian host cells to express the specified gene segment. If the number of lysine residues in the peptide core is large and the length is long, it is better to use recombinant protein technology. This is because when the length of the polypeptide becomes longer, the probability of error using the solid-state synthesis method increases, and its purity and / or yield decreases. To use a bacterial or mammalian host cell to generate a polypeptide nucleus, a padding sequence of 2 to 20 amino acid residues can be inserted between two K residues. In addition, since AHA and HPG are not natural amino acids that can be encoded by genetic codons, the N-terminal or C-terminal residues of these recombinant polypeptides may be cysteine. After the recombinant protein is expressed and purified, the terminal cysteine residue and the short diplex cross-linker can be reacted; The SH group reacts with cis-butene diimino group, and the other end bears an alkynyl group, an azide group, a tetrazine group or a high-stress alkynyl group.

Compared with conventional amino acids (such as glycine and serine residues) to synthesize polypeptide core, there are fewer steps to synthesize polypeptide core using PEGylated amino acid. In addition, PEGylated amino acids of different lengths (i.e., those with different EG repeat units) can be used. On the one hand, they can provide flexibility and water solubility, and on the other hand, they can also be used in adjacent lysine residues. Provide spacing between bases. In addition to PEGylated amino acids, the central core may also contain artificial amino acids, such as D-configuration amino acids, homoamino acids, N-methylamino acids, and the like. In a better case, a central nucleus can be constructed using PEGylated amino acids with different lengths of polyethylene glycol (PEG), as the PEG moieties contained in the amine molecule can provide conformable It is flexible and provides proper spacing between the groups used for joining, and can also increase its water solubility; in addition, PEG is generally less immunogenic. The method of synthesizing a central core with a PEGylated amino acid is similar to that of a conventional polypeptide.

If necessary, the central core of the present invention may further carry an acetamidine group to protect the amino acid residue at the N-terminus, thereby improving its stability.

As can be understood, the number of linking arms attached to the central core mainly depends on the number of lysine residues contained in the central core. Since the central core of the present invention contains at least two lysine residues, the junction unit may include a plurality of linking arms.

Refer to Figure 1A. As shown in the figure, the junction unit 10A includes a central core 11a with one HPG (G ^HP ) residue and four lysine (K) residues, with a filling sequence in between (“·· "..." means). The stuffing sequence between HPG residues and K residues or between any two K residues may be the same or different amino acid sequences. In this embodiment, the four connecting arms 20a-20d are connected to each of the lysine residues through a amide bond formed between the NHS group and the amino group of the lysine residue. It can be understood that there may be some common features among the various joint units described in this specification, so some or all of the features described for the joint unit 10A described above or each joint unit described below may also be applicable to the embodiments proposed below. Unless it is clearly contrary to the context of a particular embodiment. For brevity, these common features may not be repeated in the following.

FIG. 1B illustrates a bonding unit 10B according to another embodiment of the present disclosure. The central core 11b has one cysteine (C) residue and six lysine (K) residues, and these residues are separated by padding sequences. In the present embodiment, the joining unit 10B includes six connecting arms 20a-20f connected to each of the lysine residues. According to an embodiment of the present disclosure, the connecting arm is a PEG chain having 2 to 20 EG repeat units.

The bonding unit 1B is different from the bonding unit 10A in FIG. 1A in that the bonding unit 1B further includes a coupling arm 60. As described above, the coupling arm 60 can be formed by using a PEG chain having a cis-butene diimide group at one end and a special functional group at the other end. In this way, the coupling arm 60 can be connected to the cysteine residue of the central core 11b through a sulfide-cis-butene diamidide reaction. In this embodiment, the special functional group carried on the free end of the coupling arm 60 is a tetrazinyl group 72. According to an embodiment of the present disclosure, the coupling arm is a PEG chain having 2 to 12 EG repeat units.

When the effector element needs to be released at the target site, a cleavable bond can be added to the connecting arm; the cleavable bond can be cleaved by acid / base hydrolysis, reduction / oxidation, or enzyme action. For example, NHS-PEG _2-20 -SS-cis-butenediamidine can be used as the coupling arm described here, which is a cleavable PEG chain in which the SS disulfide bond is slowly reduced The NHS group can be used to join with the amino group of the central core to connect the PEG chain to the central core. In addition, an azide group, an alkynyl group, a tetrazinyl group, or a high-stress alkynyl group may be used instead of the cis-butenyldiimino group at the free end of the linking arm. According to some embodiments of the present disclosure, the linking arm is a PEG chain with 2 to 20 EG repeating units, and its free end (ie, the end not connected to the central core) has a double sulfur bond. Next, referring to FIG. 1C, the five connecting arms 21a-21f shown in the figure are respectively connected to the K residues of the central core 11b. These connecting arms are PEG chains with a disulfide bond at the free end.

According to an embodiment of the present invention, the linking arm of the K residue attached to the central core bears a functional group at the free end (i.e., cis butylene diimide, NHS group, azido, alkynyl, tetrazinyl or High stress alkynyl). In the preferred case, when the free end of the linker is azide, alkynyl or cyclooctynyl, the amino acid residue at the N- or C-terminus of the central core is a cysteine residue, and The free end of the coupling arm is tetrazinyl or cyclooctenyl. Alternatively, when the free end of the linker is tetrazinyl or cyclooctenyl, the amino acid residue at the N- or C-terminus of the central core bears an azide or alkynyl group; or it is at the central core N- Or the C-terminal amino acid residue is a cysteine residue, and the free end of the coupling arm bears an azide group, an alkynyl group or a cyclooctynyl group.

Depending on the functional group located on the free end of the linking arm (ie, cis-butenylimide, NHS, azide, alkynyl, tetrazinyl, or high-stress alkynyl), it can be designed with the corresponding function Functional element (such as a target element, an effect element, or an element for improving pharmacokinetic properties) such that the functional element can be connected to the free end of the connecting arm by any of the following chemical reactions: (1 ) Formation of amidamine bond: In this case, the free end of the linking arm has an NHS group, and the functional element has an amino group; (2) sulfhydryl-cis-butenediamidine reaction: in this case, the linking arm's The free end has a cis-butene diimide group and the functional element has a sulfhydryl group; (3) monovalent copper-catalyzed azide-alkyne hydroxy cycloaddition (copper (I) -catalyzed azide-alkyne cycloaddition; CuAAC, or "link" reaction for short): one of the free end of the linking arm and the functional element bears an azide group, while the other bears an alkynyl group; the CuAAC reaction is shown in reaction formula 1; (4) Inverse electron demand Diels–Alder (iEDDA) One of the free end of the link arm and the functional element bears a tetrazinyl group and the other bears a cyclooctenyl group; the iEDDA reaction is shown in Reaction Scheme 2; or (5) a stress-promoted azide-alkynyl hydroxyl group Strain-promoted azide-alkyne click chemistry (SPAAC): one of the free ends and functional elements of the linking arm bears an azide group and the other bears a cyclooctynyl group; the SPAAC reaction is as shown in Equation 3 Show.

CuAAC reaction will produce 1,5-disubstituted 1,2,3-triazole. The reaction selectivity between alkynyl and azide is extremely high, and natural biomolecules do not contain alkynyl and azide. Furthermore, the above reaction is fast and insensitive to pH. It is known that in addition to using monovalent copper (such as copper (I) bromide or copper iodide) to catalyze the linking reaction, it is preferred to use a mixture of divalent copper and a reducing agent (such as sodium ascorbate) in the reaction. Monovalent copper is generated in situ in the system. Alternatively, a copper-free reaction can be used to connect the second element to the N- or C-terminus of the central core. This reaction can utilize a cyclopentylcyclopentadienyl ruthenium chloride complex. ) As a catalyst to catalyze the azide-alkyne cycloaddition reaction.

＜＜ 反應式 2 iEDDA 反應 ＞＞

＜＜ 反應式 3 SPAAC 反應 ＞＞

For convenience of explanation, the functional element connected to the connecting arm is referred to as a first element. As can be appreciated, the number of first elements that the junction unit of the present invention can carry depends on the number of K residues in the central core (and therefore, on the number of linking arms). Therefore, a person having ordinary knowledge in the technical field to which the present invention belongs can adjust the number of the first elements in the bonding unit to achieve a desired target or therapeutic effect. << Reaction formula 1 CuAAC reaction >>

＜＜ Reaction formula 2 iEDDA reaction ＞＞

＜ Reaction formula 3 SPAAC reaction ＞＞

As shown in FIG. 1D, the exemplary bonding unit 10D includes a first element. Except for the features described below, the structure shown in Fig. 1D is similar to that in Fig. 1B. First, the central core 11d has five K residues, and therefore five link arms 20a-20e are connected to these K residues, respectively. Second, the bonding unit 10D has five first elements 30a-30e, which are connected to each of the connecting arms 20a-20e, respectively. As described below, the tetrazinyl group 72 is added as needed so that the junction unit can be coupled to an additional functional element or another molecular construct.

Figure 1E is an alternative embodiment. The bonding unit 10E shown is similar in structure to the bonding unit 1D, except that the component connection ends of each connection arm 21a-21e (that is, the components connected to the first components 30a-30e) One end) carries a double sulfur bond.

Alternatively, the bonding unit proposed here further includes a plurality of linking arms with a functional group at one end (ie, cis butylene diimide, NHS group, azido, alkynyl, tetrazine or high stress Alkynyl), with NHS or maleimide at the other end. The connection between the linking arm and the linking arm is similar to the connection between the first element and the linking arm. By selecting the corresponding functional group, the linking arm and the linking arm can form a hydrazine bond or pass sulfide-cis Butene diamidine, CuAAC, iEDDA, or SPAAC react and couple to each other. Therefore, the linking arm connected to the linking arm has an NHS group or a cis-butenylimide group at the free end (or the element linking end; that is, the end not connected to the linking arm); after that, the first element and The connecting ends of the linking arms form a amide bond or undergo a sulfhydryl-cis butane diimide reaction to connect to it.

Referring next to Figure 1F, the linker shown is linked to the K residue of the central core 11d in a similar manner to Figure 1D. Compared with the joint unit 10D, the joint unit 10F further includes an engagement arm 25 that is connected to the connection arm 22 through a SPAAC reaction. Then, the first element 30 and the linking arm 25 can be connected to each other by forming a amide bond or through a sulfhydryl-maleimide reaction. The diamond-shaped point 90 shown in FIG. 1F represents a chemical bond formed by the SPAAC reaction between the connecting arm 22 and the connecting arm 25.

According to some embodiments of the present disclosure, the adaptor arm is a PEG chain with 2 to 20 EG repeat units. Alternatively, the adaptor arm is a PEG chain with 2 to 20 EG repeat units, and its element-linking end (ie, the free end not connected to the linker arm) has a double sulfur bond.

In an experimental example, the adaptor arm has three EG repeat units, and the free end of the adaptor arm (also referred to as the element connection end) has a double sulfur bond. In this example, when treated with a reducing agent, the first element connected to the connecting end of the engagement arm element can be effectively released by the bonding unit proposed here.

According to some preferred embodiments of the present disclosure, the first element may be fingolimod, fingolimod phosphate, interferon-β or integrin-α4, β-amyloid, viral or bacterial protein Specific single-stranded variable fragment (scFv).

Non-limiting viral proteins include: F protein of respiratory fusion virus (RSV), gp120 protein of human immunodeficiency virus type I (HIV-1), hemagglutinin A (HA) protein of influenza A virus and giant cells Glycoproteins of the virus.

Examples of bacterial proteins include, but are not limited to, endotoxins of Gram-negative bacteria, surface antigens of Clostridium difficile, staphic acid of Staphylococcus aureus, anthrax toxin of Bacillus anthracis, or type I or Type II Shiga toxin.

In order to further increase a desired effect (eg, therapeutic effect), the bonding unit of the present invention may include a second element in addition to the first element. For example, the second element may be a target element or an effect element. In an optional embodiment of the present disclosure, the first element is an effect element, and the second element may be another effect element, and the two may act cumulatively or multiply, or may act independently. In another example, the first and second elements may play different properties; for example, the first element is a target element, and the second element is an effect element, and vice versa. Alternatively, the first element is an effector element, and the second element can improve the pharmacokinetic properties of the junction unit, such as water solubility, clearance rate, half-life, and bioavailability. When selecting a specific first element and / or a second element included in the bonding unit (or multi-arm bonding object), the desired application needs to be considered. Embodiments of these functional elements are discussed in section (iii), section I below.

Structurally, the second element may be attached to the N- or C-terminal azide, alkynyl, tetrazinyl, or high-stress alkynyl group of the central core. Specifically, when necessary, the second element may be complexed with a short PEG chain (preferably having 2 to 12 EG repeat units), and then connected to an N- or C-terminated azide Amino or alkynyl amino acid residues (such as AHA residues or HPG residues). Alternatively, when needed, the second element can be complexed with the short PEG chain and then connected to the coupling arm of the central core.

According to certain embodiments of the present disclosure, the N- or C-terminus of the central core is an amino acid residue having an azide group (such as an AHA residue); and therefore, a second element bearing an alkynyl group may be Connected to the N- or C-terminus of the central core by CuAAC reaction. According to other embodiments of the present disclosure, the N- or C-terminus of the central core is an amino acid residue having an alkynyl group (such as an HPG residue); and the second element having an azide group can pass through CuAAC The reaction is connected to the N- or C-terminus of the central core.

FIG. 1G illustrates another bonding unit 10G according to an embodiment of the present invention, which carries a plurality of first components and a second component. In this embodiment, the central core 11c includes one HPG (G ^HP ) residue and five lysine (K) residues. The five connecting arms 20a-20e are connected to the five K residues of the central core 11c, and the five first elements 30a-30e are connected to each of the connecting arms 20a-20e through the sulfhydryl-cis butane diimine reaction. . In addition to the first element, the bonding unit 10G further includes a second element 50 connected to one end of the short PEG chain 62. Before complexing with the central core 11c, the other end of the short PEG chain 62 has an azide group. In this way, the azide group can perform a CuAAC reaction with the alkynyl-containing HPG residue, so that the second element 50 is connected to the central core 11c. The solid dot 40 shown in Figure 1G represents the chemical bond formed between the HPG residue and the azide group due to the CuAAC reaction.

Alternatively, the second element is connected to the central core through the coupling arm. According to some embodiments of the present disclosure, the coupling arm is provided with a tetrazinyl group, and thus can be efficiently connected to the second element with TCO through the iEDDA reaction. According to other embodiments of the present disclosure, the coupling arm has a TCO group, and the second element may have a tetrazine group, so the two can be connected to each other through an iEDDA reaction. Compared to the terminal alkynyl group, the activation energy of the strained cyclooctenyl group used in the iEDDA reaction is significantly lower, and therefore no additional catalyst is required for the iEDDA reaction.

Referring next to Figure 1H, the central core 11d of the junction unit 10H includes a cysteine (C) residue and five lysine (K) residues at the N-terminus. As shown in Figure 1H, the five link arms 20a-20e are connected to the five K residues of the central core 11d, and the five first elements 30a-30e are reacted through the sulfhydryl-maleimide Instead, they are connected to five connecting arms 20a-20e, respectively. The cysteine residue is connected to the coupling arm 60; before the second element is connected, the free end of the coupling arm 60 bears a tetrazine group or a TCO group. In this embodiment, the second element 50 may be connected to the short PEG chain 62 with the corresponding TCO or tetrazine group, and then connected to the coupling arm 60 through the iEDDA reaction. The ellipse 70 shown in FIG. 1H represents a chemical bond generated by the iEDDA reaction between the coupling arm 60 and the short PEG chain 62.

According to other embodiments of the present disclosure, the coupling arm has an azide group before the second element is bonded; when the second element and the free end have a cyclooctyne group (such as DBCO, DIFO, BCN, or DICO group), When the PEG chain is connected, the coupling arm and the second element can be connected through the SPAAC reaction, and vice versa.

Referring next to FIG. 1I, the structure of the bonding unit 10I is similar to that of the bonding unit 10H in FIG. 1H, except that the coupling arm 60 has an azide group or a cyclooctyne group (such as DBCO, DIFO, BCN, or DICO group). Instead of tetrazine or TCO. Therefore, the second element 50 must be connected to a short PEG chain 62 with a corresponding cyclooctyne group (such as DBCO, DIFO, BCN, or DICO) or an azide group, so that the second element 50 and the coupling arm 60 can react through SPAAC. While connected. The diamond-shaped dot 90 shown in FIG. 11I represents a chemical bond formed by the SPAAC reaction between the coupling arm 60 and the short PEG chain 62.

Scheme 4 is an exemplary scheme for preparing a junction unit of the present invention. In step 1, a central core is first prepared, the amino acid sequence of which contains (GSK) ₃ and the C-terminus is an L-azidohomogenine (AHA) residue. In step 2, by forming a amide bond between the NHS group and the amino group, the three connecting arms are respectively connected to the lysine (K) residue of the central core, and the connecting arm connected to the central core has a cis Butene difluorenimine (Mal) group. In step 3, three anti-A antigens scFv (scFv α A) are connected to each linking arm through a sulfhydryl-maleimide reaction, where scFv α A is the first element. At the same time, in step 4, an anti-B antigen scFv (scFv α B) is connected to a short PEG chain with a DBCO group at the free end. The short PEG chain has 4 EG repeat units. Here, scFv α B is the first Two components. Finally, in step 5, a second element is connected to the AHA residue of the central core through a SPAAC reaction. << Reaction formula 4 to prepare two different methods of joining scFv unit is connected via a connection arm and C- terminal amino acid residues >>

Scheme 5 illustrates another exemplary process for preparing a bonding unit of the present invention. In step 1, a central core is first prepared, the amino acid sequence of which contains (KX _aa ) ₃ and the cysteine residue at the C-terminus. In step 2, a PEG chain with a tetramethylene group at one end and a tetrazinyl group at the other end (as a coupling arm) is connected to the cysteine through a sulfhydryl-maleimide reaction Amino acid residues. Thereafter, in step 3, the three linking arms are each connected to the lysine (K) residue of the central core. Next, as shown in step 4, three anti-A antigens scFv (scFv α A) are connected to each linking arm through a sulfhydryl-maleimide reaction, where scFv α A is the first element. . At the same time, in step 5, an anti-B antigen scFv (scFv α B) is connected to a short PEG chain with a TCO group at the free end. The short PEG chain has 3 EG repeat units. Here, scFv α B is the first Two components. Finally, in step 6, the second element is connected to the coupling arm through the iEDDA reaction. ＜< Reaction method 5 for preparing a bonding unit for connecting two different scFvs through a connecting arm and a coupling arm >＞

Pegylation is a method of attaching or linking a PEG chain to a molecule (eg, a drug or a protein). PEGylation can improve a number of important pharmacological properties, such as increasing water solubility, extending the life cycle, and reducing proteolytic degradation. According to an embodiment of the present disclosure, the second element is a PEG chain having a molecular weight of about 20,000 to 50,000 Daltons.

FIG. 1J illustrates another exemplary junction unit 10J, in which five first elements 30 are connected to lysine residues through the connection arm 20, and the HPG (G ^HP ) residues of the central core 11e pass through the CuAAC reaction. Linked to PEG chain 80. The solid dot 40 shown in Figure 1J represents the chemical bond formed between the HPG residue and the PEG chain 80 due to the CuAAC reaction.

FIG. 1K is another embodiment of the present disclosure. The N-terminus of the central core 11d of the junction unit 10K is a cysteine residue connected to the coupling arm 60. The PEG chain 80 can be efficiently connected to the coupling arm 60 through the iEDDA reaction. The elliptic point 70 of the bonding unit 10K represents the chemical bond formed between the coupling arm 60 and the PEG chain 80 due to the iEDDA reaction.

FIG. 1L is another embodiment of the bonding unit of the present invention. The structure of the bonding unit 10L shown in FIG. 1J is similar to that of the bonding unit 10J in FIG. 1J, except that the PEG chain 80 is connected to the coupling arm 60 through a SPAAC reaction. The diamond-shaped dot 90 shown in FIG. 1L represents a chemical bond formed by the SPAAC reaction between the coupling arm 60 and the PEG chain 80.

According to some embodiments of the present disclosure, in addition to the first and second elements, the bonding unit of the present invention further includes a third element. In such embodiments, the N- or C-terminus of the central core is an amino acid residue bearing an azide or alkynyl group, and the other end is a cysteine residue. The lysine residues in the central core are connected to the linking arms, respectively, and the free end of each linking arm bears a cis-butene diimide group; the cysteine residues in the central core and the free end bear Tetrazinyl or high-stress alkynyl coupling arms are connected. In this way, the first element can be connected to the connecting arm through the sulfhydryl-maleimide reaction, and the second element can be connected to the coupling arm through the iEDDA reaction. In addition, the third element is connected to a terminal amino acid residue having an azide group or an alkynyl group through a CuAAC reaction or a SPAAC reaction.

Referring next to the joining unit 10M in FIG. 1M, the N-terminus of the central core 11f is an HPG (G ^HP ) residue, and the C-terminus is a cysteine residue. The connecting arm 20 and the coupling arm 60 are respectively connected to the lysine (K) residue and the cysteine (C) residue of the central core 11f. In addition, the five first elements 30 are connected to five connecting arms 20, the second element (ie, the PEG chain) 80 is connected to the coupling arm 60, and the third element 50 is connected to the HPG residue through the short PEG chain 62. The solid dot 40 represents the chemical bond formed between the HPG residue and the short PEG chain 62 due to the CuAAC reaction; and the oval dot 70 represents the chemical bond formed between the coupling arm 60 and the PEG chain 80 due to the iEDDA reaction.

FIG. 1N illustrates another embodiment of the present disclosure; the structure of the junction unit 10N is similar to the junction unit 10M shown in FIG. 1M, except that the short PEG chain 62 is connected to HPG residues through a SPAAC reaction Rather than through iEDDA. The diamond-shaped point 90 in FIG. 1N represents the chemical bond formed between the short PEG chain 62 and the HPG residue due to the SPAAC reaction.

In a preferred embodiment of the present disclosure, the free end of the linking arm is provided with a cis-butenediimide group to react with the first element having a sulfhydryl group through the sulfhyde-cisbutenediimide While connected. In addition, one end of the polypeptide core may be a cysteine residue or an amino acid residue bearing an azide or alkynyl group for attachment to a coupling arm to connect a second element.

Those skilled in the art to which the present invention pertains can conceive of various modifications to the above structure. For example, functional groups (such as azide, alkynyl, tetrazinyl, or high-stress alkynyl) other than cis butylene diimide can be used at the free end of the linking arm to pass through CuAAC, iEDDA, or SPAAC React to connect the first element. In addition, the cysteine residue (or an amino acid residue bearing an azide or alkynyl group) may be located at a position other than the N- or C-terminus of the polypeptide core. What is more, two or more of the above residues can be added to the polypeptide core for joining multiple coupling arms and further connecting multiple second elements.

(ii) Compound cores for multiarm conjugates

In addition to the junction units described in section (i) of section one of this disclosure, another junction unit is also disclosed herein that uses a compound (rather than a polypeptide) as the central core. Specifically, the compound may be benzene-1,3,5-triamine, 2- (aminemethyl) -2-methylpropane-1,3-diamine, tri (2-amineethyl) amine , Benzene-1,2,4,5-tetramine, 3,3 ', 5,5'-tetraamine-1,1'-biphenyl, tetra (2-amineethyl) methane, tetra (ethylamine) Hydrazine, N, N, N ', N',-tetrakis (amineethyl) ethylenediamine, benzene-1,2,3,4,5,6-hexaamine, 1-N, 1-N, 3- N, 3-N, 5-N, 5-N-hexa (methylamine) -benzene-1,3,5-triamine, 1-N, 1-N, 2-N, 2-N, 4-N , 4-N, 5-N, 5-N, -octa (methylamine) -benzene-1,2,4,5-triamine, benzene-1,2,3,4,5,6-hexaamine or N, N-di [(1-amino-3,3-diamineethyl) pentyl] methanediamine. Each of these compounds carries at least three amino groups in an equal or symmetrical configuration. Therefore, when one of the amino groups of a compound is compounded with a coupling arm, all the molecules formed by the compound have the same configuration.

The compounds described here are similar to the junction units described in section (i) of the first section of this disclosure, and each contain a plurality of amino groups, and therefore, a plurality of PEG chains bearing an NHS group can pass through the amino group and the NHS The amine bond formed between the two groups is connected to the compound core; the PEG chain connected in this way is the linking arm described in this application, and the free end of the linking arm carries a functional group (such as NHS group, cis Butenediamidino, azido, alkynyl, tetrazinyl, cyclooctenyl or cyclooctynyl). At the same time, the compound core has at least one amino group connected to another PEG chain. The PEG chain has an NHS group at one end and a functional group (eg, azide, alkynyl, tetrazinyl, cyclooctenyl) at the other end. Or cyclooctynyl); the compound core and this type of PEG chain are also connected through a fluorene bond, and this PEG chain is called a coupling arm, and its free end bears the above-mentioned special functional group.

Therefore, the first element can be connected to the connecting arm by: (1) forming a fluorene bond between the two; (2) through the sulfhydryl-cis butylene diimine reaction; (3) through CuAAC reaction; (4) reaction through iEDDA; or (5) reaction through SPAAC. At the same time, the second element can be connected to the coupling arm through a CuAAC reaction, an iEDDA reaction, or a SPAAC reaction.

According to certain embodiments of the present disclosure, the linking arm is a PEG chain having 2 to 20 EG repeating units; in a preferred case, the linking arm is a PEG chain having 2 to 20 EG repeating units and at its free The end (ie, the end that is not connected to the central core) carries a double sulfur bond. The coupling arm is a PEG chain with 2 to 12 EG repeat units. In one embodiment, the connecting arm and the coupling arm each have 12 EG repeating units, wherein before the coupling, one end of the coupling arm is an NHS group and the other end is an alkynyl group.

According to an alternative embodiment of the present disclosure, the engaging unit further includes a plurality of engaging arms, respectively connected to the respective connecting arms. Thereafter, a plurality of first elements are respectively connected to the engagement arms. In one embodiment, the adaptor arm is a PEG chain having 2 to 20 EG repeat units. In another embodiment, the adaptor arm is a PEG chain having 2 to 20 EG repeat units, and has a double sulfur bond at the connecting end of the element that is not connected to the linker arm.

Reaction formulas 6 and 7 respectively show the connection relationship between the central compound core and the linking arm and between the central compound core and the coupling arm, where NHS stands for NHS esters, Mal stands for maleimide diimide, and azide stands Azide and alkyne represent alkynyl.

＜＜ 反應式 7 將分別帶有順丁烯二醯亞胺基與炔基的連接臂與耦合臂連接到中心核 ＞＞

The compound as the central core should have polysymmetric and amino groups in the same direction for the following reasons: When one of the amine groups is used to connect a diplex bonding arm (that is, N-hydroxysuccinimide (NHS) ester Group with an alkynyl, azide, tetrazinyl or high-stress alkynyl group at the other end, a homogeneous product can be obtained (that is, the central core connected to the coupling arm, and the coupling arm has an alkynyl, azide Group, tetrazinyl group or high stress alkynyl group), so it can be easily purified. This product can then be used to prepare a multi-armed junction unit, and the remaining amino groups are connected to a connecting arm with a cis-butenylimide group or other coupling group at the free end. If a compound having multiple amino groups is an asymmetric compound, the product obtained after the compound is bonded to the duplex / arm coupling arm is not a homogeneous product. ＜< Reaction formula 6 connects a linking arm and a coupling arm each having a cis-butenediamidoimide group and an azide group to the central core ＞>

<<< Reaction formula 7 connects a linking arm and a coupling arm each having a cis-butene difluorenimide group and an alkynyl group to the central core >>

The above symmetric compound can be further modified so that it can be connected with more linking arms / coupling arms. For example, tetrakis (2-aminoethyl) methane is a compound that can be synthesized from a general compound or commercially available. Such a compound can be used to construct a junction unit that connects four connecting arms / coupling arms. Tetrakis (2-aminoethyl) methane and bis (sulfosuccinimidyl) suberate were subjected to a condensation reaction, and the resulting product was N, N-di [(1-amino- 3,3-diamineethyl) pentyl] methanediamine. This product has two tetra (2-amineethyl) methane units and can be used to connect six connecting / coupling arms. The bonding units described here are provided with three, four, and six connecting arms / coupling arms, respectively. For constructing a joint with a target / effect molecule, the number of the connecting arms / coupling arms should be quite sufficient.

It will be understood that the number of linking arms and / or coupling arms and the number of elements connected to them all depend on the number of amino groups contained in the central core. In some preferred embodiments, the number of connecting arms / coupling arms and the number of components connected thereto is 1 to 7.

Referring to FIG. 2, benzene-1,2,4,5-tetramine with four amino groups is shown; three amino groups are connected to the connecting arm 20 and the other amino group is connected to the coupling arm 60, The free end of the coupling arm 60 is provided with an azide group. After that, the three first elements 30 are connected to the three connecting arms 20 through the sulfhydryl-maleimide reaction, and one second element 50 is connected to the coupling arm 60 through the CuAAC reaction. The solid dots 40 shown in FIG. 2 represent chemical bonds formed by the CuAAC reaction between the coupling arm 60 and the second element 50.

(iii) Functional elements for dobby joints

When the engagement unit (or multi-armed junction) contains only the first element without the second and / or third element, the first element is an effector element that can exert a therapeutic effect in the patient. On the other hand, when the bonding unit proposed here includes other elements other than the first element, at least one of the two elements should be an effect element, and the other can be another effect element or a standard element Or elements that can improve the pharmacokinetic properties of the junction unit (eg, solubility, clearance, half-life, and bioavailability). For example, the joint unit can be provided with two different effect elements, an effect element and a target element or an element that enhances pharmacokinetic properties, two different target elements and one effect element, two different effect elements, and one Target element; or an element that can carry a target element, an effect element, and an improved pharmacokinetic characteristic of the junction unit.

According to certain embodiments of the present disclosure, the target element or effector element is fingolimod, fingolimod phosphate, interferon-β or is specific for integrin-α4, β-amyloid, viral or bacterial proteins Single-stranded variable fragment (scFv).

Examples of viral proteins include, but are not limited to: F protein of respiratory fusion virus (RSV), gp120 protein of human immunodeficiency virus type I (HIV-1), hemagglutinin A (HA) protein of influenza A virus, and Glycoproteins of cytomegalovirus.

Illustrative examples of bacterial proteins include: endotoxins of Gram-negative bacteria, surface antigens of Clostridium difficile, staphic acid of Staphylococcus aureus, anthrax toxin of Bacillus anthracis, or type I or II of E. coli Shiga toxin.

A long PEG chain with a molecular weight of 20,000 to 50,000 daltons is an element that improves the pharmacokinetic properties of the junction unit.

The following examples illustrate the functional elements contained in multi-arm conjugates that can be used to treat certain diseases.

In the treatment of CNS diseases (such as multiple sclerosis), exemplary junction units may use fingolimod, fingolimod phosphate, interferon-β, or scFv specific to integrin-α4 as the first element ( Effect element). In the treatment of Alzheimer's disease, the junction unit proposed here can use a scFv specific to β-amyloid as the first element (effector element). Where necessary, the junction unit used to treat multiple sclerosis, Alzheimer's disease, or other CNS diseases may further include a second element, such as a scFv specific to the transferrin receptor as the target element.

Similarly, in the treatment of infections caused by viruses or bacteria, scFv specific to the virus or bacteria protein can be used as the target element (first element). In these cases, the bonding unit may include a second element added as needed, such as an scFv specific to CD32 or CD16b as an effect element.

(iv) Use of dobby joints

The present disclosure also relates to methods for treating various diseases using appropriate junction units. Generally, the method includes the step of administering an effective amount of an engagement unit according to an embodiment of the present disclosure to an individual in need of treatment.

Compared with existing therapeutic constructs, the bonding unit described in the first paragraph and the above has at least the following advantages: (1) The number of functional elements can be adjusted according to needs and / or uses. According to the needs of different applications, the bonding unit of the present invention may include two types of components (ie, first and second components) or three types of components (ie, first, second, and third components). The above requirements include diseases to be treated, The administration method of the junction unit, the binding force and affinity of the antibody carried by the junction unit, and the like. For example, when the engagement unit is to be directly delivered to a specific tissue / organ (for example, treating the eye), only one element can be used as an effect element without the need to add the target element. However, when the junction unit is delivered via a peripheral route of administration, such as by oral, gastrointestinal, nasal, topical or mucosal administration, or by intramuscular, intravenous or intraperitoneal injection, the junction unit of the present invention needs to include the target element In order to specifically mark the joint unit to the lesion site; in addition, the joint unit should also contain effect elements in order to exert a therapeutic effect at the lesion site. In order to improve the target or therapeutic effect or enhance the stability of the bonding unit, the bonding unit of the present invention may further include a third element; the third element may be a second target element, a second effect element, or a PEG chain . (2) The first element exists in the form of a bundle. As mentioned above, the number of first elements depends on the number of lysine residues contained in the central core. If the number of lysine residues in the central core is 2 to 15, each junction unit may carry at least two first elements. Therefore, compared to traditional therapeutic constructs that can only carry a single molecule (such as a single cytotoxic drug or antibody molecule), the junction unit of the present invention can simultaneously provide more functional elements (whether the target element or the effector element) , Which can greatly improve the efficacy.

In certain therapeutic applications, it may be desirable to use a single target or effect element. For example, a single target element can be used to avoid undesired side effects caused by the strong binding of the target element; when the scFv used has a relatively high affinity for the target antigen, and when the target antigen is a normal cell (not the target The above considerations are even more important when using cell surface antigens on infected cells. In one embodiment, when scFv specific to CD3 or CD16a is used to recruit T cells or NK cells to destroy the target cells (such as thyroid cells in patients with Graves' disease), it is preferable to use a single serving of CD3. Or CD16a-specific scFv, which can avoid the adverse side effects caused by CD3 or CD16a cross-linking. Similarly, when scFv specific to CD3 or CD16b is used to recruit phagocytic neutrophils and macrophages to remove virus or bacterial particles or products bound to antibodies, a single scFv may be used. In addition, when a scFv specific to the transferrin receptor is used to carry effector drug molecules to the BBB to treat CNS diseases, it is preferred to use a single scFv specific to the transferrin receptor. In another example, you may want to add a single serving of long-chain PEG to improve the pharmacokinetics of the junction unit; using two or more long-chain PEG chains may entangle the PEG chains and affect the target or effect. The bonding force of the components.

Experimental example

Experimental example 1 ： Synthesis of peptides as peptide cores 1 ( Serial number: 18) Peptide 2 ( Serial number: 27) And peptides 3 ( Serial number: 19) And the cysteine residue SH Cis-butene diimide -PEG _3- Trans - Cyclooctene (TCO) complex

A similar approach was used to process synthetic peptides 1, 2 and 3 (Chinapeptide Inc., Shanghai, China). Individual peptides were dissolved in 100 mM sodium phosphate buffer (pH 7.0; containing 50 mM NaCl and 5 mM EDTA) to a final concentration of 2 mM. The dissolved polypeptide was reduced with 1 mM tris (2-carboxyethyl) phosphine (TCEP), and the reaction temperature was 25 ° C for 2 hours. Polypeptide and maleimide-PEG ₃ -TCO (Conju-probe Inc.) were mixed at a molar ratio of 1 / 7.5, and reacted at pH 7.0 and 25 ° C for 18 hours to make half maleic SH group (PEI) -PEG ₃ -TCO composite cysteine residues and is connected with the functional group TCO. Reverse phase HPLC was used to purify the TCO-polypeptide complex; a Supelco C18 column (250 mm X 10 mm; 5 μm) was used, and the mobile phase was acetonitrile and 0.1% trifluoroacetic acid with a linear gradient of 0% to 100% acetonitrile, 30 Minutes, flow rate 1.0 mL / min, column temperature 25 ° C.

The synthesized TCO-polypeptide was confirmed by MALDI-TOF mass spectrometry (shown below). Mass spectrometry analysis was performed at the core facility of the Institute of Molecular Biology, Academia Sinica (Taipei, Taiwan). Measurements were performed with a Bruker Autoflex III MALDI-TOF / TOF mass spectrometer (Bruker Daltonics, Bremen, Germany).

The molecular weight of the synthetic TCO-polypeptide 1 (shown below) is 2,078.9 Daltons.

The molecular weight of the synthetic TCO-polypeptide 2 (shown below) is 2,020.09 Daltons.

所述TCO-多肽3 (如下所示)的分子量為3,381.85道耳頓。

The molecular weight of the TCO-polypeptide 3 (shown below) is 3,381.85 Daltons.

Experimental example 2 ： Synthesis of peptides as peptide cores 1 And the cysteine residue SH Cis-butene diimide -PEG _4- Tetrazine complex

Synthetic peptide 1 (Chinapeptide Inc., Shanghai, China) was dissolved in 100 mM sodium phosphate buffer (pH 7.0; containing 50 mM NaCl and 5 mM EDTA) to a final concentration of 2 mM. The dissolved peptide was reduced with 1 mM TCEP, and the reaction temperature was 25 ° C for 2 hours. Polypeptide and maleimide-PEG ₄ -tetrazine (Conju-probe Inc.) were mixed at a ratio of 1/5, and reacted at pH 7, 4 ° C for 18 hours to make cysteine The SH group of the amino acid residue is complexed with maleimide-PEG ₄ -tetrazine with a functional linking group tetrazine. Reverse phase HPLC was used to purify the tetrazine-polypeptide complex; a Supelco C18 column (250 mm X 10 mm; 5 μm) was used, and the mobile phase was acetonitrile and 0.1% trifluoroacetic acid with a linear gradient of 0% to 100% acetonitrile, 30 minutes, flow rate 1.0 mL / min, column temperature 25 ° C.

The molecular weight of the synthetic tetrazine-polypeptide 1 (shown below) is 2,185.2 Daltons.

Experimental example 3 : Will be used as the connecting arm NHS-PEG _12- Maleimide and TCO- Peptide 1 Amino compound

Three PEG ₁₂ -cis-butene diimine linking arms were attached to the polypeptide core TCO-polypeptide 1. Cross-linker NHS-PEG ₁₂ -succinimidyl-imide (succinimidyl-[(N-cis-butenediimino-propylamido) -dodecyl glycol] ester [(N-maleimido-propionamido) -dodecaethyleneglycol] ester) was purchased from Thermo Fisher Scientific Inc. (Waltham, USA). Conjugation was performed according to the manufacturer's instructions. In short, peptides with lysine residues were dissolved In complex buffer phosphate buffered saline (PBS; pH 7.5) at a concentration of 100 mM. NHS-PEG ₁₂ -maleimide crosslinker was added to the dissolved peptide to a final concentration of 1 mM (compared to 0.1 mM peptide solution, 10-fold excess Molar number). The reaction mixture was allowed to react at room temperature for 18 hours. The cis-butenediimine-PEG ₁₂ -TCO-polypeptide 1 complex was purified by reverse phase HPLC; using a Supelco C18 tube Column (250 mm X 4.6 mm; 5 μm), mobile phase using acetonitrile and 0.1% trifluoroacetic acid, linear gradient 0% to 100% acetonitrile, 30 minutes, flow rate 1.0 mL / min, column temperature 25 ° C.

MALDI-TOF mass spectrometry analysis was performed to confirm the synthesized maleimide-PEG ₁₂ -TCO-polypeptide 1 complex.

The molecular weight of the synthetic maleimide-PEG ₁₂ -TCO-polypeptide 1 complex is 4,332 Daltons. As shown below, the maleimide-PEG ₁₂ -TCO-polypeptide 1 complex is a binding unit using a polypeptide core, which is connected with a coupling arm with a TCO group and three with a maleimide PEG imine PEG linker.

Experimental example 4 : Will be used as the connecting arm NHS-PEG _12- Maleimide and tetrazine - Peptide 1 Amino compound

Three PEG ₁₂ -cis-butenedifluorene imine linking arms were attached to the polypeptide core tetrazine-polypeptide 1. Cross-linker NHS-PEG ₁₂ -cis-butenediamidoimide (succinimidylimide-[(N-cis-butenediamidoimino-propanamido) -dodecyl glycol] ester (purchased from Thermo Fisher Scientific Inc., Waltham, USA). The ligation was performed according to the manufacturer's instructions. Briefly, peptides with lysine residues were dissolved in complex buffer phosphate buffered saline (PBS; pH 7.5), Concentration 100 mM. Add NHS-PEG ₁₂ -cis-butenediamidoimine cross-linker to the dissolved peptide to a final concentration of 1 mM (10 times more moles than the 0.1 mM peptide solution). Make the reaction mixture at The reaction was carried out at room temperature for 18 hours. The cis-butenediimine-PEG ₁₂ -tetrazine-polypeptide 1 complex was purified by reversed-phase HPLC; a Supelco C18 column (250 mm X 4.6 mm; 5 μm) was used for the mobile phase. Acetonitrile and 0.1% trifluoroacetic acid, a linear gradient of 0% to 100% acetonitrile, 30 minutes, a flow rate of 1.0 mL / min, and a column temperature of 25 ° C.

The synthetic maleimide-PEG ₁₂ -tetrazine-polypeptide 1 complex (shown below) is a junction unit using a polypeptide core, which is connected with a coupling arm with a tetrazine group and three A PEG linker with a cis-butene diamido group. The MALDI-TOF analysis results in Figure 2 indicate that the molecular weight of this construct is 4,461 Daltons.

Experimental example 5 : Fingolimod and fingolimod phosphate molecules and NHS-PEG ₅ -NHS Crosslinker complex

Fingolimod was purchased from Biotang Inc. (Lexington, USA), while fingolimod phosphate was purchased from KM3 Scientific Corporation (New Taipei City, Taiwan). The NH ₂ group of the fingolimod molecule is reacted with the homo-bifunctional cross-linker NHS-PEG ₅ -NHS, as shown in Reaction Scheme 8. Fingolimod and NHS-PEG ₅ -NHS were each dissolved in 100% DMSO, and the final concentrations of the two were 10 mM and 250 mM, respectively. 6% (v / v) alkaline sodium phosphate buffer (pH 12.7) was added to the fingolimod solution and incubated for 10 minutes to activate fingolimod's NH ₂ group. NHS-PEG ₅ -NHS cross-linker was added to the dissolved fingolimod solution to a final concentration of 30 mM (compared to a 10 mM fingolimod solution, the molar number was 3 times greater). The reaction mixture was incubated at room temperature for 3 hours. ＜< Composition of fingolimod molecule of reaction formula 8 with NHS-PEG ₅ -NHS crosslinker >＞

Fingolimod phosphate and NHS-PEG ₅ -NHS were each dissolved in 100% DMSO, and the final concentrations of the two were 5 mM and 250 mM, respectively. The NHS-PEG ₅ -NHS cross-linker was added to the dissolved fingolimod phosphate solution to a final concentration of 15 mM (compared to a 5 mM fingolimod solution with a 3-fold excess in mole number). The reaction mixture was incubated at room temperature for 3 hours; then 18% (v / v) acidic sodium phosphate buffer (pH = 0.88) was added to quench the reaction. The solvent was evaporated under vacuum.

NHS-PEG ₅ -fingolimod complex and NHS-PEG ₅ -fingolimod complex phosphate were dissolved in 30% acetonitrile and then purified by reversed-phase HPLC; Supelco C18 column (250 mm X 4.6 mm; 5 μm), using acetonitrile and 0.1% trifluoroacetic acid as the mobile phase, a linear gradient of 30% to 100% acetonitrile, 30 minutes, a flow rate of 1.0 mL / min, and a column temperature of 25 ° C.

According to the analysis result in FIG. 3, the molecular weight of the synthetic NHS-PEG ₅ -fingolimod complex (as shown in Reaction Formula 8) is 725.41 Daltons.

The molecular weight of the synthetic NHS-PEG ₅ -fingolimod complex phosphate (shown below) is 803.3 Daltons.

Experimental example 6 : Fingolimod molecule and NHS-SS-PEG _3- Azide linker compound

The NH ₂ group of the fingolimod molecule and the hetero-bifunctional cleavable cross-linker NHS-SS-PEG ₃ -azido (Conju-probe Inc.) were reacted at a molar ratio of 1: 3. The product was purified by HPLC azido -PEG ₃ -SS- fingolimod, to remove excess, unreacted fingolimod molecule. The method of recombination and purification is similar to that described in the examples above.

Molecular weight of the azide synthesis of complex fingolimod -PEG ₃ -SS- (shown below) is 629.33 Daltons.

Experimental example 7 : Will azide -PEG ₃ -SS- Fingolimod complex molecule with NHS-PEG _4- Dibenzocyclooctyne (DBCO) Crosslinker complex

The azide-PEG ₃ -SS-fingolimod complex molecule and the NHS-PEG ₄ -DBCO cross-linker were each dissolved in 100% DMSO, and the final concentrations of the two were 10 mM and 250 mM, respectively. In 100 mM sodium phosphate buffer (pH 7.5), 5 μl of the NHS-PEG ₄ -DBCO cross-linker was added to 400 μl of the dissolved azide-PEG ₃ -SS-fingolimod complex solution, and finally Moore ratio of 1: 3.2 ([NHS-PEG 4 -DBCO]: [ -PEG ₃ -SS- fingolimod azide compound]). The reaction mixture was incubated at room temperature for 3 hours.

The molecular weight of the synthetic NHS-PEG ₄ -PEG ₃ -SS-fingolimod complex (shown below) is 1,278.61 Daltons. Two isotopic peaks can be seen in the MS spectrum, with positions of 1,279.64 and 1,280.635, corresponding to [M + H + 1] ⁺ and [M + H + 2] ⁺ respectively.

Experimental example 8 :will NHS-PEG _5- Fingolimod complex molecule and TCO- Peptide 2 versus 3 complex

TCO-polypeptide 2 was dissolved in 100 mM sodium phosphate buffer (pH 7.5) at a concentration of 20 mM; NHS-PEG ₅ -fingolimod complex was dissolved in 100% DMSO at a concentration of 50 mM. TCO-polypeptide 2 and NHS-PEG ₅ -fingolimod complex were mixed in 100% DMSO at a mole ratio of 1/42 and incubated at room temperature for 3 hours. Next, in 100% DMSO, additional TCO-polypeptide 2 was added to the reaction solution so that the final molar ratio was 1: 13.5 ([TCO-polypeptide 2]: [NHS PEG ₅ -fingolimod complex]) . The mixture was further incubated at room temperature for 3 hours. The data in Figure 4 show that the molecular weight of the drug-loaded bundle formed by TCO-polypeptide 2 and fingolimod is 5,069 daltons.

TCO-polypeptide 3 was dissolved in 100 mM sodium phosphate buffer (pH 7.5) at a concentration of 10 mM; NHS-PEG ₅ -fingolimod complex was dissolved in 100% DMSO at a concentration of 50 mM. In 100% DMSO, TCO-polypeptide 2 was mixed with NHS-PEG ₅ -fingolimod complex at a mole ratio of 1/42 and reacted overnight at room temperature. The data in Figure 5 shows that the molecular weight of the drug-loaded bundle formed by TCO-polypeptide 3 and fingolimod is 9,479 daltons, which means that fingolimod molecules are indeed complexed to the TCO-polypeptide 3 junction unit.

The synthesized drug-loaded bundle (shown below) consists of a junction unit with a free TCO functional group and a group of five fingolimod molecules.

The second drug-loaded bundle (shown below) consists of a junction unit with a free TCO functional group and a group of ten fingolimod molecules.

Experimental example 9 : will NHS-PEG _5- Fingolimod complex phosphate molecule and TCO- Peptide 2 complex

TCO-polypeptide 2 and NHS-PEG ₅ -fingolimod complex phosphate were mixed in 100 mM sodium phosphate buffer (pH 7.5) at a molar ratio of 1/42 and reacted at room temperature for 3 hours . Mass spectrometry analysis showed that the molecular weight of the drug-loaded bundle formed by TCO-polypeptide 2 and fingolimod phosphate was 5,379.16 Daltons (Figure 6).

The synthesized drug-loaded bundle (shown below) consists of a junction unit with a free TCO functional group and a set of five fingolimod phosphate molecules (p fingolimod as an effector element).

Experimental example 10 :will NHS -PEG ₄ -PEG ₃ -SS- Fingolimod complex molecule and TCO- Peptide 2 complex

The five NHS-PEG ₄ -PEG ₃ -SS- fingolimod complex molecule attached to the polypeptide TCO- 2. The NHS-PEG ₄ -PEG ₃ -SS-fingolimod complex molecule is complexed to the NH ₂ group of the TCO-polypeptide 2 lysine residue using a method similar to that described in the above example. The product was confirmed by MALDI-TOF mass spectrometry.

The synthesized drug-loaded bundle (shown below) has a molecular weight of 7,815 Daltons; it consists of a junction unit with a free TCO functional group and a set of five fingolimod molecules.

Experimental example 11 :use HEK293F Over-expression system to prepare recombinant humans CD32a Extracellular domain

The gene sequence encoding the target protein was placed in the pG1K expression cassette. The extracellular part of human CD32a is a recombinant protein with a histidine tag, and its amino acid sequence is shown in SEQ ID NO: 28. Recombinant human CD32a extracellular domain was expressed in FreeStyle 293F suspension culture cell expression system and medium (Invitrogen, Carlsbad, USA). FreeStyle 293F cells were seeded in 600 mL of Expi293F performance medium at a density of 2.0 x 10 ⁶ viable cells per milliliter, and maintained for 18 to 24 hours before transfection to ensure that the cells were in a dividing state during transfection. For transfection, place 96 mL of culture medium with 1.0 × 10 ⁷ cells in a 2-L Ehrlich flask and add linear polyethylenimine (linear polyethylenimine; average molecular weight 25 kDa; purchased from Polysciences, Warrington) , USA) as transfection reagent. After transfection, the transfected cells were incubated for 4 hours at 37 ° C with an orbital shaker (125 rpm); afterwards, the cell density was adjusted to 2.5 × 10 ⁶ cells / ml with fresh medium and incubated 4 to 5 day. The culture supernatant was collected and the protein in the culture medium was purified using nickel affinity chromatography. Figure 7 is the result of SDS-PAGE analysis of purified human CD32a extracellular domain protein.

Experimental example 12 :use HEK293F Preparation of recombinant human transferrin by overexpression system -1 Receptor (TfR) Extracellular domain

The gene sequence encoding the target protein was placed in the pG1K expression cassette. The expressed human TfR1 extracellular domain is a recombinant protein with a histidine tag, and its amino acid sequence is shown in SEQ ID NO: 29. Recombinant human TfR1 extracellular domain was expressed in FreeStyle 293F suspension culture cell expression system and medium. FreeStyle 293F cells were seeded in 600 mL of Expi293F performance medium at a density of 1.0 x 10 ⁶ live cells per milliliter, and maintained for 18 to 24 hours before transfection to ensure that the cells were in a dividing state during transfection. For transfection, a 96 mL medium with 1.0 × 10 ⁷ cells was placed in a 2-L Ehrlich flask, and a linear polyethyleneimine with an average molecular weight of 25 kDa was added as a transfection reagent. After transfection, the transfected cells were incubated for 4 hours at 37 ° C with an orbital shaker (125 rpm); afterwards, the cell density was adjusted to 2.5 × 10 ⁶ cells / ml with fresh medium and incubated 4 to 5 day. The culture supernatant was collected and the protein in the culture medium was purified using nickel affinity chromatography. Figure 7 is the result of SDS-PAGE analysis of purified human TfR1 extracellular domain protein.

Experimental example 13 :use Expi293F Over-performance system preparation RSV protein F Monoclonal Antibody, Monoclonal Antibody to Endotoxin CD32a Extracellular domain specific mAb scFv

The V _L and V _H lines of the scFv specific to RSV protein F are derived from the monoclonal antibody palivizumab; the V _L and V _H lines of the scFv specific to endotoxin are from the monoclonal antibody WN1 222-5 (US Patent No. 5,858,728) ); The V _L and V _H lines of the scFv specific to the extracellular domain of CD32a are from MDE-8 (US Patent Application Publication US 2007/0253958). The scFv derived from the above antibody is specially designed to carry a flexible conjugate (GGGGSGGGGS) and a terminal cysteine residue at the C-terminus. The thiol group of the cysteine residue can be bonded to the maleimide group at the free end of the linking arm of each bonding unit. When preparing scFvs such as monoclonal antibodies specific to RSV protein F, monoclonal antibodies specific to endotoxin, and monoclonal antibodies specific to the extracellular domain of CD32a, DNA sequences encoding the three antibodies V _L and V _H were used and performed. Codon optimization. A DNA sequence encoding the following sequence was synthesized: V _L -GSTSGSGKPGSGEGSTKG-V _H- (GGGGS) ₂ -C. The scFv amino acid sequences of the monoclonal antibodies specific to RSV protein F, the monoclonal antibodies specific to endotoxin, and the monoclonal antibodies specific to the extracellular domain of CD32a prepared in the experimental examples of the present invention are as follows: 30 to 32.

When the mammalian expression system is used to prepare scFv proteins, the Expi293F ™ cell line-based overexpression system is used to prepare the scFv protein. This system uses the ExpiFectamine ™ 293 transfection kit (Life Technologies, Carlsbad, USA), which contains Expi293F ™ cell lines, the cationic lipid-based ExpiFectamine ™ 293 reagent, ExpiFectamine ™ 293 transfection enhancers 1 and 2, and the performance system Medium (Gibco, New York, USA).

The scFv-encoding sequence was placed in the pG1K expression cassette. Expi293F cells were seeded in Expi293F performance medium at a density of 2.0 x 10 ⁶ viable cells per milliliter, and maintained for 18 to 24 hours before transfection to ensure that the cells were in a dividing state during transfection. For transfection, place 255 ml of culture medium with 7.5 × 10 ⁸ cells in a 2-L Ehrlich flask and add ExpiFectamine ™ 293 transfection reagent. After transfection, the transfected cells were incubated for 16 to 18 hours at 37 ° C with an orbital shaker (125 rpm); after that, ExpiFectamine ™ 293 transfection enhancer 1 and enhancer 2 were added to the flask and continued to grow 5 to 6 days. The culture supernatant was collected and the scFv protein in the culture medium was purified using protein L affinity chromatography.

Figures 9A and 9B are the results of SDS-PAGE and ELISA analysis of purified scFv against RSV protein F-specific monoclonal antibodies, and Figures 9C and 9D are the results of SDS-PAGE and ELISA analysis of purified scFv against endotoxin-specific monoclonal antibodies. Figures 9E and 9F are the results of SDS-PAGE and ELISA analysis of purified scFv of CD32a extracellular domain-specific monoclonal antibody, respectively. A 96-well ELISA plate (Greiner Bio-one) was coated with 5 μg / ml of RSV protein F, 10 μg / ml of endotoxin and 5 μg / ml of the extracellular domain of CD32a. HRP-labeled protein L was used to detect and purify scFv at a ratio of 1: 5000.

ELISA analysis showed that each purified scFv protein can specifically bind to its respective antigen (RSV protein F, endotoxin or CD32a extracellular domain). The above analysis used adalimumab scFv (anti-TNF-α scFv) served as a negative control group.

Experimental example 14 :use Expi293F Over-performance system preparation TfR1 Extracellular domain specific mAb and pair β- Amyloid-specific mAb scFv

V _L and V _H based on the specificity of the scFv TfR1 from the extracellular domain of a monoclonal antibody OX26; V _L and V _H based on the specificity of the scFv β- amyloid protein from horses daclizumab bar. The scFv derived from the above antibody is specially designed to carry a flexible conjugate (GGGGSGGGGS) and a terminal cysteine residue at the C-terminus. The thiol group of the cysteine residue can be bonded to the maleimide group at the free end of the linking arm of each bonding unit. When the preparation of the extracellular domain TfR1 specific monoclonal antibody and scFv of β- amyloid-specific monoclonal antibodies, using two DNA sequences encoding the above antibody V _L and V _H and codon optimized. A DNA sequence encoding the following sequence was synthesized: V _L -GSTSGSGKPGSGEGSTKG-V _H- (GGGGS) ₂ -C. The amino acid sequences of scFv specific to the TfR1 extracellular domain-specific monoclonal antibody and β-amyloid-specific monoclonal antibody prepared in the experimental examples of the present invention are shown as sequence numbers: 33 and 34, respectively.

When the mammalian expression system is used to prepare scFv proteins, the Expi293F ™ cell line-based overexpression system is used to prepare the scFv protein. This system uses the ExpiFectamine ™ 293 transfection kit (Life Technologies, Carlsbad, USA), which contains Expi293F ™ cell lines, the cationic lipid-based ExpiFectamine ™ 293 reagent, ExpiFectamine ™ 293 transfection enhancers 1 and 2, and the performance system Medium (Gibco, New York, USA).

The scFv-encoding sequence was placed in the pG1K expression cassette. Expi293F cells were seeded in Expi293F performance medium at a density of 2.0 x 10 ⁶ viable cells per milliliter, and maintained for 18 to 24 hours before transfection to ensure that the cells were in a dividing state during transfection. For transfection, place 255 ml of culture medium with 7.5 × 10 ⁸ cells in a 2-L Ehrlich flask and add ExpiFectamine ™ 293 transfection reagent. After transfection, the transfected cells were incubated for 16 to 18 hours at 37 ° C with an orbital shaker (125 rpm); after that, ExpiFectamine ™ 293 transfection enhancer 1 and enhancer 2 were added to the flask and continued to grow 5 to 6 days. The culture supernatant was collected and the scFv protein in the culture medium was purified using protein L affinity chromatography. Figures 10A and 10B are SDS-PAGE and ELISA analysis results of purified scFv of TfR1 extracellular domain-specific monoclonal antibody, respectively. Figures 10C and 10D are SDS-PAGE and SDS-PAGE analysis of purified scFv of β-amyloid-specific monoclonal antibody, respectively. ELISA analysis results. The 96-well ELISA plate (Greiner Bio-one) was coated with 5 μg / ml TfR1 extracellular domain and 5 μg / ml β-amyloid. HRP-labeled protein L was used to detect and purify scFv at a ratio of 1: 5000.

ELISA analysis showed that each purified scFv protein could specifically bind to its respective antigen (TfR1 extracellular domain or β-amyloid protein). The above analysis used HRP-labeled protein L alone as a negative control group.

Experimental example 15 : Construction and Choice for Humans CD32a Extracellular domain-specific phage - Present scFv

By agreement between the two parties, a phage strain with scFv specific for the extracellular domain of human CD32a was obtained by Dr. Yang Ansui's laboratory at the Genomics Research Center of the Central Academy of Sciences (Taipei, Taiwan). The framework sequence of the GH2 scFv antibody library was derived from the G6 anti-VEGF Fab (protein library code: 2FJG), and was cloned between the restriction sites SfiI and NotI of the phage vector pCANTAB5E (GE Healthcare). The plastid vector carries an ampicillin resistance gene, a lacZ promoter, a pelB leader sequence that facilitates secretion of the scFv fragment into the culture supernatant, and an E-tag for detection. The template is scFv V _H and V _L domains based on diverse oligonucleotide site-directed mutagenesis (oligonucleotide-directed mutagenesis) techniques; the variable region of each of the three CDR diversification simultaneously. The scFv antibody library with more than 10 ⁹ selected strains was used to screen on the extracellular domain of CD32a.

The Maxisorp 96-well plate (Nunc) was coated with recombinant CD32a protein (1 μg / 100 μL PBS per well) to pan out anti-CD32a antibodies. In short, when coating human CD32a on a well plate, the coating solution can be injected into the well and shaken at room temperature for 2 hours. The CD32a-coated wells were then treated with a blocking buffer at room temperature. The blocking buffer was dissolved in PBST with 5% skim milk (phosphate buffered buffer with 0.1% tween-20) for 1 hour. . After the recombinant phage in the blocking buffer was diluted to a concentration of 8 × ^{10 11} colony-forming unit (CFU) per ml, it was added to a CD32a-coated well dish and gently shaken for 1 hour. The well plate was then vigorously washed ten times with PBST, and then washed six times with PBS to remove phage that had not specifically bound. The bound phage was eluted with 0.1 M HCl / glycine buffer (pH 2.2), and the eluate was immediately neutralized with 2 M Tris-base buffer (pH 9.0). E. coli strain ER2738 (OD ₆₀₀ = ~ 0.6) was used for phage infection at 37 ° C for 30 minutes; ampicillin was used for 30 minutes to remove uninfected E. coli. After ampicillin treatment, a helper bacteriophage M13KO7 with kanamycin resistance was added and the incubation was continued for 1 hour. Phages that can be rescued by helper phages were selected from E. coli cultures and shaken vigorously at 37 ° C overnight in an environment with concomycin to amplify the selected phages. The amplified phage was precipitated in PEG / NaCl and then resuspended in PBS for the next selection-amplification cycle. The above selection-amplification cycle was repeated to perform three consecutive pannings on the CD32a extracellular domain.

After serial dilution of phage-infected ER2738 colonies, the number and phage titer were calculated to obtain the output titer / ml (CFU / ml) per milliliter after each panning round. After three rounds of panning, the phage output titer increased by more than 1000 times from 1.6E + 04 CFU / well to 2.2E + 07 CFU / well. Figure 11A shows the phage export / import titer ratio for each round. The Y-axis shows the phage output / input titer ratio for each panning round. After three rounds of panning, the proportion of positive colonies increased significantly. Compared with the first round, the output / input titer ratio of the third round increased by 100 times, and the combined colonies gradually became the dominant group in the antibody library.

In a general selection procedure, after three rounds of antigen panning on ELISA well plates coated with human CD32a, about 80% of the bound phage particles can specifically bind to the coated CD32a in the ELISA analysis.

Experimental example 16 : For humans CD32a Extracellular domain-specific phage - Present scFv Single colony ELISA analysis

E. coli strain ER2738 was infected with a single colony phage, and selected scFv genes were obtained from the phage of each phage; 2YT culture medium (containing 16 g / L tryptic protein, 10 g / L yeast) was added to the deep wells. The extract and 5 g / L NaCl; pH 7.0) and 100 μg / ml ampicillin were shaken at 37 ° C to cultivate these E. coli to the mid-log phase. When the OD ₆₀₀ of the culture solution reached 1.0, IPTG was added to a final concentration of 1 μg / ml. The plate was incubated overnight at 37 ° C with vigorous shaking; thereafter, the plate was centrifuged at 4,000 g for 15 minutes at 4 ° C.

The soluble scFv binding test was performed using ELISA. Briefly, Maxisorp 96-well plate (Nunc) was coated with the extracellular domain of CD32a (0.5 μg / 100 μl PBS per well) or human transferrin-1 receptor (as a negative control group) and shaken at 4 ^o C 18 hours. After treating with 300 μl of blocking buffer for 1 hour, 100 μl of the supernatant containing soluble scFv and 100 μl of blocking buffer were mixed, and then this mixture was added to the coated well plate for another hour. Goat anti-E-tag antibody (complexed with HRP, 1: 4000, catalog number AB19400, Abcam) was added to the well plate for 1 hour. TMB matrix (50 μl per well) was added to the well plate, and after 1N HCl (50 μl per well) was added to stop the reaction, the absorbance at 450 nm was measured.

After three rounds of panning, a total of 192 phages were selected for analysis in this experimental example. OD ₄₅₀ of which the difference (differential _of OD ₄₅₀₎ after 12 and CD32a scFv binding is greater than 10, and further this coding sequence of a given gene sequence of scFv 12, and identify six different DNA sequences. Fig. 11B shows the results of an ELISA analysis of the scFv strain 22D1. The scFv strain 22D1 and human CD32a bind to an OD ₄₅₀ of 0.8, and its amino acid sequence is shown in SEQ ID NO: 35.

Experimental example 17 : Construction and Choice for Humans TfR1 Extracellular domain-specific phage - Present scFv

By agreement between the two parties, a phage strain with scFv specific for the extracellular domain of human TfR1 was obtained by Dr. Yang Ansui's laboratory at the Genomics Research Center of the Chinese Academy of Sciences (Taipei, Taiwan). The framework sequence of the GH2 scFv antibody library is derived from the G6 anti-VEGF Fab (protein library code: 2FJG), and it is cloned between the restriction sites SfiI and NotI of the phage vector pCANTAB5E (GE Healthcare). There is an ampicillin resistance gene, a lacZ promoter, a pelB guide sequence that helps secrete scFv fragments into the culture supernatant, and an E-tag for detection. Diversify the V _H and V _L domains of scFv template based on oligonucleoside site-directed mutagenesis technology; simultaneously diversify the three CDRs in each variable region. The scFv antibody library with more than 10 ⁹ clones was used to screen on the TfR1 extracellular domain.

The Maxisorp 96-well plate (Nunc) was coated with recombinant TfR1 protein (1 μg / 100 μL PBS per well) to pan out anti-TfR1 antibodies. In short, when coating human TfR1 on a well plate, the coating solution can be injected into the wells and shaken at room temperature for 2 hours. TfR1-coated wells were then treated with a blocking buffer at room temperature. The blocking buffer was dissolved in PBST with 5% skim milk (phosphate buffered buffer with 0.1% tween-20) for 1 hour. . After the recombinant phage in the blocking buffer was diluted to a concentration of 8 × ^{10 11} CFU / mL, it was added to a TfR1-coated well plate and gently shaken for 1 hour. The well plate was then vigorously washed ten times with PBST, and then washed six times with PBS to remove phage that had not specifically bound. The bound phage was eluted with 0.1 M HCl / glycine buffer (pH 2.2), and the eluate was immediately neutralized with 2 M Tris-base buffer (pH 9.0). E. coli strain ER2738 (OD ₆₀₀ = ~ 0.6) was used for phage infection at 37 ° C for 30 minutes; ampicillin was used for 30 minutes to remove uninfected E. coli. After the ampicillin treatment, the co-mycin-resistant helper phage M13KO7 was added and the incubation was continued for 1 hour. Phages that can be rescued by helper phages were selected from E. coli cultures and shaken vigorously at 37 ° C overnight in an environment with concomycin to amplify the selected phages. The amplified phage was precipitated in PEG / NaCl and then resuspended in PBS for the next selection-amplification cycle. The above selection-amplification cycle was repeated to perform three consecutive pannings on the TfR1 extracellular domain.

After serial dilution of phage-infected ER2738 colonies, the number and phage titer were calculated to obtain the output titer / ml (CFU / ml) per milliliter after each panning round. After three rounds of panning, the phage output titer increased by more than 10,000 times from 3.74E + 03 CFU / well to 1.5E + 08 CFU / well. Figure 12A shows the phage export / import titer ratio for each round. The Y-axis shows the phage output / input titer ratio for each panning round. After three rounds of panning, the proportion of positive colonies increased significantly. Compared with the first round, the output / input titer ratio of the third round increased by 10,000 times, and the combined colonies gradually became the dominant group in the antibody library.

In a general selection procedure, after three rounds of antigen panning on a human TfR1-coated ELISA plate, about 80% of phage particles bound to TfR1 can specifically bind to the coated TfR1 in an ELISA .

Experimental example 18 : For humans TfR1 Extracellular domain-specific phage - Present scFv Single colony ELISA analysis

E. coli strain ER2738 was infected with a single colony phage, and selected scFv genes were obtained from the phage of each phage; 2YT culture medium (containing 16 g / L tryptic protein, 10 g / L yeast) was added to the deep wells. The extract and 5 g / L NaCl; pH 7.0) and 100 μg / ml ampicillin were shaken at 37 ° C to cultivate these E. coli to the mid-log phase. When the OD ₆₀₀ of the culture solution reached 1.0, IPTG was added to a final concentration of 1 μg / ml. The plate was incubated overnight at 37 ° C with vigorous shaking; thereafter, the plate was centrifuged at 4,000 g for 15 minutes at 4 ° C.

The soluble scFv binding test was performed using ELISA. Briefly, Maxisorp 96- well plates (Nunc) coated TfR1 extracellular domain (per well 0.5 μg / 100 μl PBS), or CD16b (as a negative control), shaken at 4 ^o C 18 h. After treating with 300 μl of blocking buffer for 1 hour, 100 μl of the supernatant containing soluble scFv and 100 μl of blocking buffer were mixed, and then this mixture was added to the coated well plate for another hour. Goat anti-E-tag antibody (complexed with HRP, 1: 4000, catalog number AB19400, Abcam) was added to the well plate for 1 hour. TMB matrix (50 μl per well) was added to the well plate, and after 1N HCl (50 μl per well) was added to stop the reaction, the absorbance at 450 nm was measured.

After three rounds of panning, a total of 192 phages were selected for analysis in this experimental example. Among them, 23 strains of scFv and TfR1 combined had an OD ₄₅₀ difference greater than 10, further sequencing the gene sequences encoding these 23 strains of scFv, and identifying 16 different DNA sequences. Figure 12B shows the results of an ELISA analysis of the scFv strain 12A1. The scFv strain 12A1 binds to human TfR1 with an OD ₄₅₀ of 1.7, and its amino acid sequence is shown in SEQ ID NO: 36.

Experimental example 19 :preparation TCO- Correct CD32a Extracellular domain specific scFv Complex

As shown in the experimental example above, a DNA sequence encoding a protein sequence number: 32 protein was synthesized and expressed. The purified scFv of the anti-CD32a monoclonal antibody and 5 mM dithiothreitol (DTT) were incubated at room temperature with gentle shaking for 4 hours to reduce the C-terminal cysteine residues to facilitate interaction with Mal-PEG ₃ -TCO (Conju-probe, Inc.) complex. Using a NAP-10 Sephadex G-25 column, the scFv protein-reducing buffer was replaced with sodium phosphate buffer (100 mM sodium phosphate (pH 7.0), 50 mM NaCl, and 5 mM EDTA). After the reduction reaction and the buffer exchange, the reactants having a molar ratio of 10: 1 ([Mal-PEG ₃ -TCO]: [scFv]) were allowed to react overnight at room temperature for recombination. Excess crosslinks were removed using a desalting column and analyzed for TCO-scFv complex products.

The MALDI-TOF mass spectrometry analysis indicated that the molecular weight of the TCO-specific CD32a scFv complex was 27,337 Daltons. The purity of TCO-sc32 specific scFv complexes was analyzed using Coomassie staining with 12% SDS-PAGE. Figures 13A and 13B show the results of ELISA and mass spectrometry analysis of TCO-sc32v-specific scFv complex, respectively, and unmodified scFv specific to CD32a was used as a positive control group. ELISA results showed that the TCO-CD32a-specific scFv complex can bind to the extracellular domain of recombinant human CD32a.

Experimental example 20 : Preparation of tetrazine - Correct TfR1 Extracellular domain specific scFv Complex

As shown in the experimental example above, the DNA sequence encoding the protein sequence number: 33 protein was synthesized and expressed. The anti-TfR1 single purified scFv and 5 mM dithiothreitol (DTT) were incubated at room temperature with gentle shaking for 4 hours to reduce the C-terminal cysteine residues to facilitate interaction with Mal -PEG ₄ -tetrazine (Conju-probe, Inc.) complex. Using a NAP-10 Sephadex G-25 column, the scFv protein-reducing buffer was replaced with sodium phosphate buffer (100 mM sodium phosphate (pH 7.0), 50 mM NaCl, and 5 mM EDTA). After the reduction reaction and the buffer exchange, the reactants having a molar ratio of 10: 1 ([Mal-PEG ₄ -tetrazine]: [scFv]) were allowed to react overnight at room temperature for complexing. Excessive cross-links were removed using a desalting column and the tetrazine-scFv complex was analyzed.

MALDI-TOF mass spectrometry analysis indicated that the molecular weight of the tetrazine-to-TfR1-specific scFv complex was 27,086 Daltons. The purity of the tetrazine-scFv complex specific to TfR1 was analyzed by Coomassie staining with 12% SDS-PAGE. Figures 14A and 14B show the results of ELISA and mass spectrometry analysis of a tetrazine-scFv-specific scFv complex, respectively, and unmodified scFv specific to TfR1 was used as a positive control group. ELISA results showed that the tetrazine-scFv complex specific for TfR1 can bind to the extracellular domain of recombinant human TfR1.

Experimental example twenty one :will Three specific for endotoxin scFv Complex to tetrazine - Peptide 1 Three maleimide -PEG ₁₂ Connecting arm

This experimental example reveals that three scFvs can be complexed to three PEG ₁₂ -maleimide diimine linking arms on the tetrazine-polypeptide 1 core. Prior to complexing with a tetrazine-polypeptide 1 core with three PEG ₁₂ -cis-butene diamidine immobilization arms, the endotoxin-specific scFv and DTT were mole-ratio 2: 1 ([DTT]: [scFv ]) Incubation at 25 ° C for 4 hours with gentle shaking to reduce the C-terminal cysteine residues. Next, using a NAP-10 Sephadex G-25 column (GE Healthcare), the reduced endotoxin-specific scFv buffer was replaced with a maleimide-SH coupling reaction buffer (100 mM sodium phosphate). (pH 7.0), 50 mM NaCl and 5 mM EDTA). After the reduction reaction and the buffer exchange, the reactants with a molar ratio of 1: 4 ([conjugate]: [protein]) were reacted at 4 ° C overnight to make them react with Tetrazine-polypeptide 1 nuclear complex of amine-PEG ₁₂ linker.

Using a particle size sieve column S75, PEG ₁₂ -cis-butenediamidine-tetrazine-polypeptide 1 complex with three endotoxin-specific scFvs was combined with free scFv and free PEG ₁₂ -cis The butene difluorene imine-tetrazine-polypeptide 1 complex and the PEG ₁₂ -cis butadiene diimide-tetrazine-polypeptide 1 complex complexed with one or two endotoxin-specific scFvs are separated from each other.

Figure 15A is the FPLC dissolution profile of the synthesized target junction unit; the retention volume is 9.5 milliliters; the synthesized target junction unit consists of a free tetrazine functional group and a set of three pairs Endotoxin-specific scFv (as the underlying element) junction unit. The product (i.e., PEG having a functional group consisting of tetrazine and a set of three endotoxin-specific scFv ₁₂ - maleic (PEI) - tetrazine - 1 polypeptide complex) was purified to elution parts (Elution In fraction), after separation with 10% SDS-PAGE, this product is indicated by the arrow on the electrophoresis strip in Figure 15B.

Experimental example twenty two : Of the joint unit MALDI-TOF Analysis; the junction unit of this target is attached to tetrazine - Peptide 1 Nuclear three Maleimide -PEG ₁₂ Endotoxin-specific three linking arms scFv

Three target endothelial-specific scFv-specific splice unit samples with three cis-butene diimine-PEG ₁₂ linking arms attached to the tetrazine-polypeptide 1 core were subjected to MALDI-TOF analysis. The experimental median molecular weights and three endotoxin-specific scFvs are consistent with the theoretical median molecular weights of the tetrazine-polypeptide 1 nuclear coupling product with three cis-butene diimine-PEG ₁₂ linking arms. According to the mass spectrometry analysis result shown in Fig. 15C, the median molecular weight of this target junction unit was 81,727 Daltons.

As shown in the figure below, the target junction unit synthesized here has a free tetrazine function and a set of three endotoxin-specific scFvs as target elements.

Experimental example twenty three : Made from tetrazine - Peptide 1 Nuclear sum with three pairs RSV F Protein-specific scFv Composing unit

Using the method described in the experimental example above, scFv was complexed with the junction unit and further analyzed after purification.

Figure 16 is the result of mass spectrometry analysis of the synthetic target junction unit. This junction unit contains a free tetrazine functional group and a set of three scFv-specific scFvs specific to the RSV F protein (as shown in the figure below). As shown in Figure 16, the molecular weight of this standard junction unit is 81,978 Daltons.

Experimental example twenty four :will Three pairs β- Amyloid-specific scFv Coupled to Three cis-butene diimines -PEG ₁₂ Connecting arm TCO- Peptide 1 nuclear

This experimental example reveals that three scFvs can be complexed to three cis-butenylimide-PEG ₁₂ linking arms on the TCO-polypeptide 1 core. Prior to complexation with a TCO-polypeptide 1 core with three cis- butadieneimine-PEG ₁₂ linking arms, scFv and DTT specific to β-amyloid were mole-ratio 2: 1 ([DTT]: [scFv]) was incubated at room temperature for 4 hours with gentle shaking to reduce the C-terminal cysteine residues. Next, using a NAP-10 Sephadex G-25 column (GE Healthcare), the reduced buffer for β-amyloid-specific scFv was replaced with a maleimide-SH coupling reaction buffer (100 mM sodium phosphate (pH 7.0), 50 mM NaCl and 5 mM EDTA). After the reduction reaction and the buffer exchange, the reactants with a molar ratio of 1: 4 ([conjugate]: [protein]) were reacted at 4 ° C overnight to make them react with TCO-polypeptide 1 nuclear complex of amine-PEG ₁₂ linker.

The reaction mixture of the aforementioned experimental example was adjusted to pH 5.0, and then added to a pre-equilibrated cation exchange column SP Sepharose FF (GE Healthcare) pre-equilibrated with (5 mM EDTA and 50 mM sodium acetate (pH 5.0). A linear gradient of 0 to 500 mM sodium chloride complexed with three male β-amyloid scFv-specific maleimide-PEG _{12 at} a flow rate of 0.5 ml per minute for 100 minutes. -TCO-polypeptide 1 complex was precipitated. Using a cation exchange column SP Sepharose FF, cis-butene diimide-PEG ₁₂ -TCO-peptide 1 complex was compounded with three scFvs specific to β-amyloid. And free scFv, free maleimide-PEG ₁₂ -TCO-polypeptide 1 complex, and maleimide-PEG complexed with one or two β-amyloid-specific scFvs _{The 12-} TCO-polypeptide 1 complexes are separated from each other. The purified product is concentrated, that is, the cis-butenylimide-PEG ₁₂ -TCO-peptide 1 complex complexed with three scFvs specific to β-amyloid, and The buffer was replaced with a linking reaction buffer (100 mM potassium phosphate, pH 7.0).

Figure 17A is the dissolution profile of the effect junction unit synthesized after performing FPLC on a cation exchange column SP Sepharose FF; the synthesized effect junction unit consists of a free TCO functional group and a set of three Binding unit of scFv (as an effector element) specific to β-amyloid. # 1 and # 2 shown in Figure 17A represent the precipitation peaks of the cis-butenylimide-PEG ₁₂ -TCO-polypeptide 1 complex with two and three β-amyloid-specific scFvs, respectively. . Purified product (ie, a maleic acid-PEG ₁₂ -TCO-polypeptide 1 complex with a free TCO functional group and a set of three β-amyloid-specific scFvs) at 8% SDS After -PAGE separation, this product was shown in Figure 17B electrophoresis.

Experimental example 25 : Of the effect junction unit MALDI-TOF Analysis; this effect junction unit is connected to TCO- Peptide 1 Nuclear three Maleimide -PEG ₁₂ Three pairs of connecting arms β- Amyloid-specific scFv

Samples of three effector junction units for β-amyloid-specific scFv with three cis-butenimidine-PEG ₁₂ linking arms attached to the TCO-polypeptide 1 core were analyzed by MALDI-TOF. The experimental median molecular weight and three β-amyloid-specific scFvs are in agreement with the theoretical median molecular weight of the TCO-polypeptide 1 nuclear coupling product with three cis-butene diimine-PEG ₁₂ linking arms. . According to the mass spectrometry results shown in Figure 17C, the median molecular weight of this effect junction unit was 87,160 channels.

As shown in the figure below, the effector junction unit synthesized here has a free TCO functional group and a set of three scFv specific to human β-amyloid as the target element.

Experimental example 26 : Preparation of a molecular construct; this molecular construct carries three pairs as target elements RSV F Protein-specific scFv And a pair of effect elements CD32a Extracellular domain specific scFv

In this experimental example, the tetrazine group of the target junction unit prepared in the above experimental example and TCO- were subjected to an iEDDA reaction to the TCO group of the scFv complex specific to the extracellular domain of CD32a, and the two were joined. Specifically, the target junction unit carries three scFvs specific to the RSV F protein and one free tetrazine group.

The tetrazine-TCO junction was performed according to the manufacturer's instructions (Jena Bioscience GmbH, Jena, Germany). In short, 100 μl of the standard junction unit (0.3 mg / ml) was added to the solution containing the effect element, and the molar ratio of the two was 1: 1.2 ([tetrazine]: [TCO]). The reaction mixture was incubated at room temperature for 1 hour. Mass spectrometry analysis of the product showed that the molecular weight of the product was 113,036 Daltons (Figure 18A).

This product (shown below) is a single junction unit molecular construct with three scFvs (as target elements) specific to the RSV F protein and one scFv (as effector element) specific to the extracellular domain of CD32a.

Experimental example 27 : Preparation of a molecular construct; this molecular construct carries three endotoxin-specific scFv And a pair of effect elements CD32a Extracellular domain specific scFv

The tetrazine group and the TCO- of the target junction unit prepared in the above experimental example were subjected to an iEDDA reaction with the TCO group of the scFv complex specific to the extracellular domain of CD32a, and the two were joined.

The tetrazine-TCO junction was performed in the manner described in the above experimental example. The resulting product (shown below) is a single junction unit molecular construct with three scFv (as a target element) specific to endotoxin and one scFv (as an effector element) specific to the extracellular domain of CD32a. The mass spectrometry results shown in Figure 18B show that the molecular weight of this molecular construct is 113,761 Daltons.

Experimental example 28 : Preparation of a molecular construct; this molecular construct carries a pair as a target element TfR1 Extracellular domain specific scFv And three pairs as effect elements β- Amyloid-specific scFv

The TCO of the target junction unit prepared in the above experimental example and the tetrazine-tetrazine group of the scFv complex specific for the extracellular domain of TfR1 were subjected to an iEDDA reaction, and the two were joined.

The tetrazine-TCO junction was performed according to the manufacturer's instructions (Jena Bioscience GmbH, Jena, Germany). Briefly, 12.6 μl of the target element (5.65 mg / ml) was added to a solution containing a junction unit with an effect element, and the mole ratio of the two was 10: 1 ([tetrazine]: [TCO]). The reaction mixture was incubated at room temperature for 3 hours. Mass spectrometry analysis of the product showed that the molecular weight of the product was 114,248 Daltons (Figure 18C).

This product (shown below) is a single junction unit molecular construct with one scFv (as a target element) specific to the extracellular domain of TfR1 and three scFv (as an effector element) specific to β-amyloid.

It should be understood that the description of the above embodiments is merely an example, and those skilled in the art to which the present invention pertains can make various modifications thereto. The above description, experimental examples, and materials have completely explained the structure and use of the exemplary embodiment of the present invention. Although the above embodiments disclose specific examples of the present invention, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention pertains should not deviate from the principles and spirit of the present invention. Various changes and modifications can be made to it, so the scope of protection of the present invention shall be defined by the scope of the accompanying patent application.

The main component symbols are as follows:
10A‧‧‧Joint Unit
11a‧‧‧Central Nuclear
20a-20d‧‧‧Connecting arm

In order to make the above and other objects, features, advantages, and embodiments of the present invention more comprehensible, the accompanying drawings are briefly described as follows.

1A to 1N are schematic diagrams of a bonding unit according to some embodiments of the present disclosure.

Figure 2 shows the results of matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of a peptide nuclear junction unit. Tetrazinyl arm and three PEG-imine-containing PEG arm.

Figure 3 shows the results of MALDI-TOF MS analysis of NHS-PEG ₅ -fingolimod complex.

Figure 4 shows the results of MALDI-TOF MS analysis of a drug-loaded bundle consisting of a junction unit with a free TCO functional group and a group of five fingolimod molecules.

Figure 5 shows the results of MALDI-TOF MS analysis of a drug-loaded bundle consisting of a junction unit with a free TCO functional group and a group of ten fingolimod molecules.

Figure 6 shows the results of MALDI-TOF MS analysis of a drug-loaded bundle consisting of a junction unit with a free TCO functional group and a group of five fingolimod phosphate molecules.

Figure 7 shows the results of SDS-PAGE analysis of purified human CD32a extracellular domain (ectodomain).

Figure 8 shows the results of SDS-PAGE analysis of purified human TfR1 extracellular domain.

Figure 9A shows the results of SDS-PAGE analysis of purified scFv specific to RSV protein F; Figure 9B shows the results of ELISA analysis of purified scFv specific to RSV protein F; Figure 9C shows SDS-PAGE of purified scFv specific to endotoxin. PAGE analysis results; Figure 9D is the ELISA analysis result of purified scFv specific to endotoxin; Figure 9E is the SDS-PAGE analysis result of purified scFv specific to CD32a extracellular domain; and Figure 9F is purified specific to CD32a extracellular domain ScFv ELISA analysis results.

Figure 10A shows the results of SDS-PAGE analysis of purified scFv specific to rat TfR1 extracellular domain; Figure 10B shows the results of ELISA analysis of purified scFv specific to rat TfR1 extracellular domain; Figure 10C shows purified purified β-like starch Results of SDS-PAGE analysis of protein-specific scFv; and Figure 10D shows the results of ELISA analysis of purified scFv specific to β-amyloid.

Figure 11A is the virus titer data of phage with scFv specific to the extracellular domain of human CD32a; and Figure 11B is the result of single colony ELISA analysis of phage-specific scFv presenting to the human CD32a extracellular domain.

Figure 12A is the virus titer data of phage with scFv specific to the human TfR1 extracellular domain; and Figure 12B is the single colony ELISA analysis result of phage-specific scFv presenting to the human TfR1 extracellular domain.

Figures 13A and 13B are the results of ELISA analysis and mass spectrometry analysis of the TCO-CD32a-specific scFv complex, respectively.

Figures 14A and 14B show the results of ELISA and mass spectrometry analysis of the tetrazine-scFv complex specific to TfR1, respectively.

FIG. 15A is a dissolution profile of the synthesized target junction unit obtained by performing FPLC with a size exclusion column S75. The synthesized target junction unit comprises a free tetrazine functional group and a group Three junction units specific to endotoxin-specific scFv (as the target element); Figures 15B and 15C are the results of SDS-PAGE analysis and mass spectrometry analysis of the target junction unit synthesized in Figure 15A, respectively.

Figure 16 is the result of mass spectrometry analysis of the synthesized target junction unit; the target junction unit is composed of a junction unit with a free tetrazine functional group and a set of three scFv (as a target element) specific to RSV protein F. composition.

Figure 17A is a dissolution profile of the synthesized target junction unit obtained by performing FPLC with a cation exchange column. The synthesized target junction unit consists of a free TCO functional group and a set of three pairs of β-like starch. Protein-specific scFv (as the target element) junction unit; Figures 17B and 17C are the SDS-PAGE analysis results and ELISA analysis results of the synthesized target junction unit described in Figure 17A, respectively.

Figure 18A is the result of mass spectrometry analysis of a single junction unit molecular construct. This single junction unit molecular construct uses three scFvs specific to endotoxin as the target element and one scFv specific to the extracellular domain of CD32a as the effector element; 18B The figure shows the result of mass spectrometry analysis of another single junction unit molecular construct with three end-toxin-specific scFvs as target elements and one CD32a extracellular domain-specific scFv as effector elements; and Figure 18C is the result of mass spectrometry analysis of another single junction unit molecular construct. This single junction unit molecular construct uses one scFv specific to the extracellular domain of TfR1 as the target element and three scFv specific to β-amyloid. Effect element.

According to the usual operation method, various features and components in the figure are not drawn to scale. The drawing method is to present the specific features and components related to the present invention in an optimal way. In addition, between different drawings, the same or similar element symbols are used to refer to similar elements / components as much as possible.

<110> Immune Lab Co., Ltd. <120> Multi-arm conjugate with peptide core and its application <130> P2950-TW <150> US 62 / 164,400 <151> 2015-05-20 <150> US / 62 / 213,012 ＜ 151 ＞ 2015-09-01 ＜ 150 ＞ US 62 / 308,349 ＜ 151 ＞ 2016-03-15 ＜ 160 ＞ 36 ＜ 170 ＞ BiSSAP 1.3 ＜ 210 ＞ 1 ＜ 211 ＞ 4 ＜ 212 ＞ PRT ＜ 213> Artificial sequence <220> <223> filler sequence-1 <400> 1 Gly Gly Gly Ser 1 <210> 2 <211> 4 <212> PRT <213> artificial sequence <220> <223> filler sequence-2 <400 ＞ 2 Gly Ser Gly Ser 1 <210> 3 <211> 4 <212> PRT <213> Artificial sequence <220> <223> filler sequence-3 <400> 3 Gly Gly Ser Gly 1 <210> 4 <211> 5 <212> PRT <213> artificial sequence <220> <223> filler sequence-4 <400> 4 Gly Ser Gly Gly Ser 1 5 <210> 5 <211> 5 <212> PRT <213> artificial sequence <220 ＞ ＜ 223 ＞ filler sequence -5 <400> 5 Ser Gly Gly Ser Gly 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial sequence <220> <223> filler sequence-6 <400> 6 Gly Gly Gly Gly Ser 1 5 <210> 7 <211> 6 <212> PRT <213> Artificial sequence <220> <223> filler sequence-7 <400> 7 Gly Gly Ser Gly Gly Ser 1 5 <210> 8 <211> 7 <212 ＞ PRT ＜ 213 ＞ Artificial sequence ＜ 220 ＞ ＜ 223 ＞ filler sequence-8 ＜ 400 ＞ 8 Gly Gly Ser Gly Gly Ser Gly 1 5 ＜ 210 ＞ 9 ＜ 211 ＞ 8 ＜ 212 ＞ PRT ＜ 213 ＞ artificial sequence 〈220〉 ＜ 223 ＞ filler sequence-9 ＜ 400 ＞ 9 Ser Gly Ser Gly Gly Ser Gly Ser 1 5 ＜ 210 ＞ 10 ＜ 211 ＞ 9 ＜ 212 ＞ PRT ＜ 213 ＞ Artificial sequence ＜ 220 ＞ ＜ 223 ＞ filler sequence-10 ＜ 400 ＞ 10 Gly Ser Gly Gly Ser Gly Ser Gly Ser 1 5 <210> 11 <211> 10 <212> PRT <213> Artificial sequence <220 ＜ 223 ＞ filler sequence-11 ＜ 400 ＞ 11 Ser Gly Gly Ser Gly Gly Ser Gly Ser Gly 1 5 10 ＜ 210 ＞ 12 ＜ 211 ＞ 11 ＜ 212 ＞ PRT ＜ 213 ＞ Artificial sequence ＜ 220 ＞ ＜ 223 ＞ filler sequence- 12 <400> 12 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser 1 5 10 <210> 13 <211> 12 <212> PRT <213> Artificial sequence <220> <223> filler sequence-13 <400> 13 Ser Gly Gly Ser Gly Gly Ser Gly Ser Gly Gly Ser 1 5 10 <210> 14 <211> 13 <212> PRT <213> Artificial sequence <220> <223> filler sequence-14 <400> 14 Gly Gly Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Gly Ser 1 5 10 <210> 15 <211> 14 <212> PRT <213> Artificial sequence <220> <223> filler sequence-15 <400> 15 Gly Gly Gly Ser Gly Ser Gly Ser Gly Ser Gly Gly Gly Ser 1 5 10 <210> 16 <211> 15 <212> PRT <213> Artificial sequence <220> <223> filler sequence-16 <400> 16 Ser Gly Ser Gly Gly Gly Gly Gly Gly Ser Gly Gly Ser Gly Ser Gly 1 5 10 15 <210> 17 <211> 16 <212> PRT <213> Artificial sequence <220> <221> MOD_RES <222> 1 <223> ACETYLATION <220> <223> polypeptide core-1 <400> 17 Cys Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Gly Ser Gly Ser Lys 1 5 10 15 <210> 18 <211> 19 <212> PRT <213> Artificial sequence <220> <221> MOD_RES <222> 1 <223> ACETYLATION <220 ＞ 223 ＞ polypeptitde core-2 ＜ 400 ＞ 18 Cys Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Gly Ser Gly Ser Lys 1 5 10 15 Gly Ser Lys ＜ 210 ＞ 19 ＜ 211 ＞ 31 ＜ 212 ＞ PRT ＜ 213 ＞ Artificial sequence <220> <221> MOD_RES ＜ 222 ＞ 1 ＜ 223 ＞ ACETYLATION ＜ 220 ＞ ＜ 223 ＞ polypeptide-3 ＜ 400 ＞ 19 Cys Gly Ser Lys Gly Ser Lys Gly Ser Lys Gly Ser Lys Gly Ser Lys 1 5 10 15 Gly Ser Lys Gly Ser Lys Gly Ser Lys Gly Ser Lys Gly Ser Lys 20 25 30 <210> 20 <211> 8 <212> PRT <213> Artificial sequence <220> <223> hapten <400> 20 Trp Ala Asp Trp Pro Gly Pro Pro 1 5 <210> 21 <211> 16 <212> PRT <213> Artificial sequence <220> <221> MOD_RES <222> 1 <223> Xaa is homopropargylglycine <220> <221> MOD_RES <222> 1 <223> ACETYLATION <220> <220> 223 ＞ polypeptide core-4 ＜ 400 ＞ 21 Xaa Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Gly Ser Gly Ser Lys 1 5 10 15 ＜ 210 ＞ 22 ＜ 211 ＞ 19 ＜ 212 ＞ PRT ＜ 213 ＞ Artificial sequence 〈220〉 ＜ 221 ＞ MOD_RES 222 ＞ 1 ＜ 223 ＞ Xaa is homopropargylglycine ＜ 220 ＞ ＜ 221 ＞ MOD_RES ＜ 222 ＞ 1 ＜ 223 ＞ ACETYLATION ＜ 220 ＞ ＜ 223 ＞ polypeptide core-5 ＜ 400 ＞ 22 Xaa Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Gly Ser Gly Ser Lys 1 5 10 15 Gly Ser Lys <210> 23 <211> 19 <212> PRT <213> Artificial sequence <220> <221> MOD_RES <222> 1 <223> Xaa is L-azidohomoalanine <220> ＜ 221 ＞ MOD_RES ＜ 222 ＞ 1 ＜ 223 ＞ ACETYLATION ＜ 220 ＞ ＜ 223 ＞ polypeptide core-6 ＜ 400 ＞ 23 Xaa Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Gly Ser Gly Ser Lys 1 5 10 15 Gly Ser Lys ＜ 210> 24 <211> 21 <212> PRT <213> Artificial sequence <220> <221> MOD_RES <222> 1 <223> Xaa is homopropargylglycine <220> <221> MOD_RES <222> 1 <223> ACETYLATION <220 ＞ ＜ 223 ＞ polypeptide core-7 ＜ 400 ＞ 24 Xaa Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Gly Ser Gly Ser Lys 1 5 10 15 Gly Ser Gly Ser Cys 20 <210> 25 <211> 7 <212> PRT <213> Artificial Sequences <220> <221> MOD_RES ＜ 222 ＞ 1 ＜ 223 ＞ ACETYLATION ＜ 220 ＞ ＜ 223 ＞ polypeptide core-8 ＜ 220 ＞ ＜ 221 ＞ MOD_RES ＜ 222 ＞ 2,4,6 ＜ 223 ＞ Xaa is PEGylated amino acid with two EG units ＜ 400 ＞ 25 Cys Xaa Lys Xaa Lys Xaa Lys 1 5 <210> 26 <211> 11 <212> PRT <213> Artificial sequence <220> <221> MOD_RES <222> 1 <223> ACETYLATION <220> <223> polypeptide core-9 ＜ 220 ＞ ＜ 221 ＞ MOD_RES ＜ 222 ＞ 2,4,6,8,10 ＜ 223 ＞ Xaa is PEGylated amino acid with six EG units ＜ 400 ＞ 26 Cys Xaa Lys Xaa Lys Xaa Lys Xaa Lys Xaa Lys 1 5 10 ＜ 210 ＞ 27 ＜ 211 ＞ 16 ＜ 212 ＞ PRT ＜ 213 ＞ Artificial sequence ＜ 220 ＞ ＜ 223 ＞ 5xdrug ＜ 400 ＞ 27 C ys Gly Ser Lys Gly Ser Lys Gly Ser Lys Gly Ser Lys Gly Ser Lys 1 5 10 15 <210> 28 <211> 666 <212> PRT <213> Artificial sequence <220> <223> human transferrin-1 receptor <400 ＞ 28 Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro Gly Glu Asp Phe 1 5 10 15 Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu Ser 20 25 30 Glu Lys Leu Asp Ser Thr Asp Phe Thr Ser Thr Ile Lys Leu Leu Asn 35 40 45 Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Asn 50 55 60 Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe Lys Leu Ser Lys 65 70 75 80 Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val Lys Asp Ser Ala 85 90 95 Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg Leu Val Tyr Leu 100 105 110 Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Tla Val 115 120 125 Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu 130 135 140 Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly 145 150 155 160 Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala 165 170 175 Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn 180 185 190 Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro 195 200 205 Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser 210 215 220 Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala 225 230 235 240 Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Asp 245 250 255 Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser Glu Ser Lys Asn 260 265 270 Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile Lys Ile Leu Asn 275 280 285 Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val 290 295 300 Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser Gly 305 310 315 320 Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met 325 330 335 Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser 340 345 350 Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu 355 360 365 Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu 370 375 380 Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro 385 390 395 400 Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn Val Lys His Pro 405 410 415 Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val 420 425 430 Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser 435 440 445 Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp Thr Asp Tyr Pro 450 455 460 Tyr Leu Gly Thr Thr M et Asp Thr Tyr Lys Glu Leu Ile Glu Arg Ile 465 470 475 480 Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln 485 490 495 Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu 500 505 510 Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr 515 520 525 Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser 530 535 540 Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe 545 550 555 560 Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp 565 570 575 Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro 580 585 590 Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr 595 600 605 Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly 610 615 620 Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp 625 630 635 640 Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile 645 650 655 Asp Asn Glu Phe His His His His His His 660 665 ＜ 210 ＞ 29 ＜ 211 ＞ 192 ＜ 212 ＞ PRT ＜ 213 ＞ Artificial Sequence ＜ 220 ＞ ＜ 223 ＞ human CD32 ＜ 400 ＞ 29 Gln Ala Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp 1 5 10 15 Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly Ala 20 25 30 Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu 35 40 45 Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn 50 55 60 Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp 65 70 75 80 Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro 85 90 95 His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser 100 105 110 Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys 115 120 125 Ser Gln Lys Phe Ser Arg Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala 130 135 140 Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr 145 150 155 160 Thr Leu P he Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro Ser 165 170 175 Met Gly Ser Ser Pro Pro Met Gly Ile Ile His His His His His His 180 185 190 ＜ 210 ＞ 30 ＜ 211 ＞ 256 ＜ 212 ＞ PRT ＜ 213 ＞ Artificial sequence ＜ 220 ＞ ＜ 223 ＞ scFv of anti-RSV mAb ＜ 400 ＞ 30 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Cys Gln Leu Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Thr 115 120 125 Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr Leu Thr 130 135 140 Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Ser 145 150 155 160 Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala 165 170 175 Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu Lys Ser 180 185 190 Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Lys 195 200 205 Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr Cys Ala Arg 210 215 220 Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr 225 230 235 240 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Cys 245 250 255 <210> 31 <211> 257 <212> PRT <213> Artificial Sequences <220> <223> scFv of anti-endotoxin mAb <400> 31 Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Thr Ile Ser Ile Thr Cys Arg Ala Ser Gln Asn Ile Asn Ile Trp 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Val Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe S er Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ile Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Gly Gln Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser 100 105 110 Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val 115 120 125 Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met 145 150 155 160 Thr Trp Val Arg Gln Ala Pro Gly Lys Ala Pro Glu Trp Leu Ala Leu 165 170 175 Ile Arg Asn Lys Arg Asn Gly Asp T hr Ala Glu Tyr Ser Ala Ser Val 180 185 190 Lys Gly Arg Phe Thr Ile Ser Arg Asp Tyr Ser Arg Ser Ile Leu His 195 200 205 Leu Gln Met Asn Ala Leu Arg Thr Glu Asp Ser Ala Thr Tyr Tyr Tyr Cys 210 215 220 Val Arg Gln Gly Arg Gly Tyr Thr Leu Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser 245 250 255 Cys ＜ 210 ＞ 32 ＜ 211 ＞ 255 ＜ 212 ＞ PRT ＜ 213 ＞ Artificial sequence ＜ 220 ＞ ＜ 223 ＞ scFv of anti-CD32a mAb ＜ 400 ＞ 32 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser 100 105 110 Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val 115 120 125 His Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val 165 170 175 Ile Trp Tyr Asp Gly Ser Asn Tyr Tyr Tyr Thr Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Asp Leu Gly Ala Ala Ala Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Cys 245 250 255 <210> 33 <211> 251 <212> PRT <213> Artificial sequence <220> <223> scFv of anti-transferrin-1 receptor mAb <400> 33 Asp Ile Val Ile Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Thr Ile Leu Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser 100 105 110 Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val 115 120 125 Gln Leu Gln Gln Pro Gly Ala Ala Leu Val Arg Pro Gl y Ala Ser Met 130 135 140 Arg Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Thr Tyr Trp Met 145 150 155 160 Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Leu Ile Gly Met 165 170 175 Ile His Pro Ser Asp Ser Glu Val Arg Leu Asn Gln Lys Phe Lys Asp 180 185 190 Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln 195 200 205 Leu Asn Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Phe Gly Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 225 230 235 240 Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Cys 245 250 ＜ 210 ＞ 34 ＜ 211 ＞ 262 ＜ 212 ＞ PRT ＜ 213 ＞ artificial sequence ＜ 220 ＞ ＜ 223 ＞ scFv of anti-beta-amyloid Bapineuzumab mAb ＜ 400 ＞ 34 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Gly Ser Thr Ser G ly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 145 150 155 160 Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 165 170 175 Glu Trp Val Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser 180 185 190 Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 195 200 205 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp 225 230 235 2 40 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Gly Gly Gly Gly Gly Ser 245 250 255 Gly Gly Gly Gly Gly Ser Cys 260 <210> 35 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> B6 scFv of anti-human TfR derived from phage display ＜ 400 ＞ 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asp Ser Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Tyr Ser Gly Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr T yr Cys Gln Gln Ser Trp Gly Phe Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Gly 100 105 110 Ser Ile Glu Gly Arg Ser Gly Gly Gly Gly Gly Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Phe Thr Ile Gly Asn Ser Ser Ile His Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Trp Pro 165 170 175 Phe Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Ser Tyr 210 215 220 Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 225 230 235 ＜ 210 ＞ 36 ＜ 211 ＞ 243 ＜ 212 ＞ PRT ＜ ＜ 213 ＞ Artificial sequence ＜ 220 ＞ ＜ 223 ＞ scFv of anti-human CD32 derived from phage display ＜ 400 ＞ 36 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Asn Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Gly Thr Ser Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Gly Phe Gly Gly Pro Met 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Ser 100 105 110 Arg Ser Ser Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asn Asn Gly Gly Ile 145 150 155 160 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 165 170 175 Ile Trp Pro Phe Gly Gly Phe Thr Ser Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Ser Trp Phe Ser Trp Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser

10A‧‧‧Joint Unit

11a‧‧‧Central Nuclear

20a-20d‧‧‧Connecting arm

Claims (32)

A joining unit includes: a central core, a plurality of connecting arms, and a coupling arm added as needed, wherein: the central core includes: (1) a first polypeptide including a plurality of lysine, K) residues, where each K residue and its next K residue are separated by a stuffing sequence, which contains glycine (G) and serine (S) residues And the number of the K residues is 2 to 15; or (2) a second polypeptide comprising the (X _aa -K) _n sequence, wherein X _aa is a PEGylated amino acid ) It has 2 to 12 ethylene glycol (EG) repeating units, and n is an integer from 2 to 15; the connecting arms are respectively connected to the K residues of the central core, and each of these connecting The free end of the arm has a cis-butenediamidino group, an azido group, an alkynyl group, a tetrazinyl group, a cyclooctenyl group, or a cyclooctynyl group; and an amino acid residue at the N- or C-terminus of the central core. Has an azide group or an alkynyl group, or an amino acid residue at the N- or C-terminus of the central core is a cysteine residue, and the sulfhydryl group of the cysteine residue is connected to the coupling arm ,its The free end of the coupling arm includes the azide, alkynyl, tetrazinyl, cyclooctenyl, or cyclooctynyl group, wherein when the free end of the linking arm is the azide group, the alkynyl group, or the In the case of a cyclooctyne group, the amino acid residue at the N- or C-terminus of the central core is a cysteine residue, and the free end of the coupling arm is a tetrazinyl group or a cyclooctenyl group; or when the When the free end of the linking arm is tetrazinyl or cyclooctenyl, the amino acid residue at the N- or C-terminus of the central core carries an azide or alkynyl group, or the N- or C- The amino acid residue at the end is a cysteine residue, and the free end of the coupling arm is an azide group, an alkynyl group or a cyclooctynyl group. The joining unit according to claim 1, wherein the sequence of the padding sequence is GS, GGS, GSG, or any sequence shown in sequence number: 1-16. The junction unit of claim 1, wherein the first polypeptide comprises a G _1-5 SK sequence of 2-15 units. The junction unit of claim 3, wherein the first polypeptide comprises a (GSK) _2-15 sequence. The joining unit according to claim 1, wherein each of the connecting arms is a polyethylene glycol (PEG) chain, which has 2 to 20 EG repeating units. The joining unit according to claim 1, wherein each of the connecting arms is a PEG chain, which has 2 to 20 EG repeating units, and has a disulfide bond at its free end. The junction unit according to claim 1, wherein the coupling arm is a PEG chain having 2 to 12 EG repeat units. The junction unit according to claim 1, wherein the amino acid residue having an azido group is L-azidohomoalanine (AHA), 4-azido-L-phenylalanine (4-azido- L-phenyl-alanine), 4-azido-D-phenylalanine, 3-azido-L-alanine, 3-azido- D-alanine (3-azido-D-alanine), 4-azido-L-homoalanine (4-azido-L-homoalanine), 4-azido-D-peralanine (4-azido-D -homoalanine), 5-azido-L-ornithine, 5-azido-D-ornithine, 6-azido-D-ornithine -6-azido-L-lysine or 6-azido-D-lysine. The joining unit according to claim 1, wherein the amino acid residue having an alkynyl group is L-homopropargylglycine (L-HPG), D-homopropargylglycine (D-homopropargylglycine (D-HPG)) or β-homopropargyl-glycine (β-HPG). The joining unit according to claim 1, wherein the cyclooctenyl group is trans-cyclooctene (TCO); and the cyclooctynyl group is dibenzocyclooctyne (DBCO), Difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), or dibenzocyclooctyne (DICO). The junction unit according to claim 1, wherein the tetrazinyl group is 1,2,3,4-tetrazine, 1,2,3,5-tetrazine or 1,2,4,5-tetrazinyl, Or its derivatives. The bonding unit according to claim 1, further comprising a plurality of first elements, which are respectively connected to the connecting arms by: forming a amide bond therebetween or through copper-catalyzed azide-alkyne Copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, strain-promoted azide-alkyne click chemistry (SPAAC) reaction or reverse electron demand Diels-Ade (inverse electron demand Diels-Alder, iEDDA) reaction. The bonding unit according to claim 12, further comprising a second element, which is connected to the central core by any of the following methods: a CuAAC reaction between the azido or alkynyl group and the second element occurs; A SPAAC reaction between the azide or cyclooctynyl group and the second element; and an iEDDA reaction between the cyclooctenyl or tetrazinyl group and the second element. The joining unit according to claim 13, wherein: the first elements are connected to the first element through forming a amine bond with the connecting arms; and the second element is connected to the central core via a CuAAC reaction. The azide or alkynyl group of the N- or C-terminal amino acid. The bonding unit according to claim 14, further comprising a third element, which is connected to the coupling arm through an iEDDA response. The bonding unit according to claim 13, wherein: the first elements are connected to the first element through forming a amine bond with the connecting arms; and the second element is connected to the central core via a SPAAC reaction. N- or C-terminal amino groups of this azide. The bonding unit according to claim 16, further comprising a third element, which is connected to the coupling arm through an iEDDA response. The joining unit according to claim 1, further comprising a plurality of linking arms, which are connected to the linking arms through a CuAAC reaction, a SPAAC reaction, or an iEDDA reaction, respectively, and a free end of each of the linking arms has a cisbutene di Hydrazone or the NHS group. The junction unit according to claim 18, wherein each of the adapter arms is a PEG chain, which has 2 to 20 EG repeat units. The junction unit according to claim 18, wherein each of the adapter arms is a PEG chain, which has 2 to 20 EG repeat units, and has a double sulfur bond at an end not connected to the connection arm. The bonding unit according to claim 18, further comprising a plurality of first elements, which are connected to the connecting arms through the following methods: via a sulfhydryl-maleimide reaction or forming a monoamine between them key. The bonding unit according to claim 21, further comprising a second element, which is connected to the central core by any of the following methods: a CuAAC reaction between the azido or alkynyl group and the second element occurs; A SPAAC reaction between the azide or cyclooctynyl group and the second element; and an iEDDA reaction between the cyclooctenyl or tetrazinyl group and the second element. The junction unit according to claim 12, wherein the first element is fingolimod, fingolimod phosphate, interferon-β, or integrin-α4, β-amyloid, viral protein, or bacterial protein A single-chain variable fragment (scFv). The junction unit according to claim 13, wherein when the first element is the fingolimod, fingolimod phosphate, interferon-β, or a specific pair of integrin-α4 or β-amyloid In the case of scFv, the second element is a scFv specific to a pair of transferrin receptors. The junction unit according to claim 13, wherein when the first element is the scFv specific to a viral protein or a bacterial protein, the second element is a pair of CD32 or CD16b-specific scFv. The junction unit according to claim 25, wherein the viral protein is F protein of respiratory syncytia virus (RSV), and gp120 protein of human immunodeficiency virus type 1 (HIV-1) 2. Hemagglutinin A (HA) protein of influenza A virus or glycoprotein of cytomegalovirus. The junction unit according to claim 25, wherein the bacterial protein is endotoxin of Gram (-) bacteria, surface antigen of Clostridium difficile , Staphylococcus aureus ( Staphylococcus aureus ), lipoteichoic acid, anthrax toxin of Bacillus anthracis , or Shiga-like toxin type II of Escherichia coli I or II). A bonding unit for preparing a medicament for treating a central nervous system (CNS) disease in an individual in need, wherein the bonding unit is as described in claim 23. The method according to claim 28, wherein the CNS disease is multiple sclerosis and the first element is the fingolimod, fingolimod phosphate, interferon-β, or integrin-α4 specific The scFv; or the CNS disease is Alzheimer's disease, and the first element is the scFv specific to β-amyloid. A bonding unit for use in the manufacture of a medicament for treating an infectious disease in an individual in need, wherein the bonding unit is as described in claim 25. The method according to claim 30, wherein the viral protein is F protein of respiratory fusion virus (RSV), gp120 protein of human immunodeficiency virus type I (HIV-1), and hemagglutinin A of influenza A virus ( HA) protein or glycoprotein of cytomegalovirus. The method according to claim 30, wherein the bacterial protein is an endotoxin of a gram-negative bacterium, a surface antigen of Clostridium difficile, a stabic acid of Staphylococcus aureus, an anthrax toxin of Bacillus anthracis, or an Type I or Type II Shiga toxins.